Bifunctional compounds

ABSTRACT

A compound having the formula: 
     
       
         
         
             
             
         
       
     
     wherein X is S, SO or SO 2 ;
 
one of R 1 , R 2 , and R 3  is O and the others of R 1 , R 2  and R 3  are independently, the same or different, CH 2 , or CR 13  wherein, R 13  is an alkyl group, an alkenyl group, an alkynyl group, a trialkylsilyl group, or —(CH 2 ) m OR 15 , wherein R 15  is an alkyl group or an aryl group and m is an integer in the range of 1 to 10, and one of R 5 , R 6 , and R 7  is O and the others of R 5 , R 6  and R 7  are independently, the same or different, CH 2 , or CR 14  wherein, R 14  is an alkyl group, an alkenyl group, an alkynyl group, a trialkylsilyl group, or —(CH 2 ) n OR 16 , wherein R 16  is an alkyl group or an aryl group and n is an integer in the range of 1 to 10;
 
R 4  and R 8  are independently, the same or different, H, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, a C 1 -C 3  alkoxy group, an aryloxy group, or —(CH 2 ) q OR 17 , wherein R 17  is an alkyl group or an aryl group and q is an integer in the range of 1 to 10, provided that R 4  is not a C 1 -C 3  alkoxy group or an aryloxy group when R 1  or R 3  is O and R 8  is not a C 1 -C 3  alkoxy group or an aryloxy group when R 5  or R 7  is O;
 
R 9 , R 10 , R 11  and R 12  are independently, the same or different, H, an alkyl group, an alkenyl group, an alkynyl group, or an aryl group.

This application claims the benefit of U.S. Provisional Application No.61/550,114, filed Oct. 21, 2011, and U.S. Provisional Application No.61/561,030, filed Nov. 17, 2011, both of which are incorporated hereinby reference in their entireties.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grants GM067082and AI068021 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND

In both the field of development of radiation counter measures for largepopulation administration as a radiation counter measure and in thefield of development of normal tissue radiation protectors for clinicalradiotherapy, interest in new small molecule modifiers of irradiationinduced cellular tissue and organ damage has gained prominence in recentyears. Reports of effective small molecule irradiation mitigators haveincluded the GS-nitroxides, triphenylphosphonium conjugated ImidazoleFatty Acids, phospho-inositol-3 kinase inhibitors, and a variety ofother small molecules which inhibit ionizing radiation inducedapoptosis. Delivery of these small molecules at 24 hours or later aftertotal body irradiation has proven effective in animal models of totalbody irradiation and in some cases, as with GS-nitroxides, has beeneffective in multiple organ specific administration protocols forprotection of the esophagus and skin from ionizing irradiation damage.

A challenge for the development of small molecule irradiation mitigatorshas been design and implementation of a non-toxic and reliable deliverysystem. Relative insolubility of many new small molecule radiationmitigators has required administration by intravenous, intra-peritoneal,or other systemic delivery systems. Delivery formulations have requiredliposomal or other solvent systems that have been unsuitable for oraladministration.

In addition, modern drug development relies on high-throughput screeningassays. Often, these trials use compound libraries stored in solutionfor periods of several months to as long as three years. DMSO 1 has beenused as the storage solvent of choice, but problems, including compounddegradation and precipitation, are frequently encountered. In one casestudy, qualitative compound precipitation was observed in 26% of testplates. Systematic studies of compound degradation in DMSO haveindicated that approximately 50% of samples degraded over a period of 12months when stored in anhydrous DMSO at ambient temperature. Compoundstorage problems are augmented by low hydrophilicity, since a largeportion of screening libraries is composed of compounds designed forenhanced membrane permeability. The trend toward lipophilic, highermolecular weight compounds results in libraries of materials with lowerintrinsic aqueous solubilities. Current estimates state that 30-50% ofcompounds in screening libraries have aqueous solubilities of less than10 μM. These lipophilic molecules are more likely to precipitate fromDMSO stock solutions, leading to erroneously low assay concentrationswhen using the DMSO stock for sample preparation. Additionally, pooraqueous solubility causes precipitation from aqueous media afterdilution of DMSO stock solutions. When compound concentrations in assaymedia fall below calculated concentrations, flawed conclusions regardingtoxicity, efficacy, or structure-activity relationships are drawn.

Aqueous dissolution of problematic compounds can be enhanced by saltformation, or chemical modification of the substrate (formation ofpro-drugs). If these methods are not applicable, complexing agents orcosolvents can be added to aid in dissolution. Some examples includecyclodextrins, dendrimers, low molecular weight PEG's (polyethyleneglycols, e.g., PEG 400), and solvents such as glycerin and NMP(A-methylpyrrolidone). Other solubilizing agents have designs based onDMSO; one example is a polymeric sulfoxide derived frompoly-L-methionine 2 (FIG. 1).

SUMMARY

Disclosed herein are compounds of formulae I-VI as described below.

Also disclosed herein are methods of treating or preventingradiation-induced damage in a subject, comprising administering to thesubject a therapeutically effective amount of at least one compound offormulae I-VI.

Also disclosed herein are methods for increasing solubility of anorganic compound in an aqueous medium, comprising including in theaqueous medium a compound that includes an oxetane moiety.

The foregoing will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Structures of sulfoxides used for compound storage or aqueoussolubility enhancement.

FIG. 2. Compounds screened in an aqueous solubility study.

FIG. 3. Solubility of quinine 8 in aqueous solutions with sulfoxide 3(Δ) and DMSO (+) as cosolvents. Each trial was run in duplicate and eachpoint represents the average of the duplicate trials. In the case ofsulfoxide 3, the pH varied from 8.7 (with no additive) to 9.4 (with a0.25 weight fraction additive). In the case of DMSO, the pH varied from8.9 (with no additive) to 9.5 (with a 0.25 weight fraction additive).

FIG. 4. Solubility of naproxen 9 in aqueous solutions with sulfoxide 3(Δ) and DMSO (+) as cosolvents. Each trial was run in duplicate and eachpoint represents the average of the duplicate trials. In the case ofsulfoxide 3, the pH varied from 4.6 (with no additive) to 4.2 (with a0.25 weight fraction additive). In DMSO, the pH varied from 4.8 to 4.4.

FIG. 5. Solubility of carbendazim 10 in aqueous solutions with sulfoxide3 (Δ) and DMSO (+) as cosolvents. Each trial was run in duplicate andeach point represents the average of the duplicate trials. In the caseof sulfoxide 3, the pH varied from 6.7 (with no additive) to 7.0 (with a0.15 weight fraction additive). In DMSO, the pH varied from 6.8 (with noadditive) to 7.4 (with a 0.25 weight fraction additive).

FIG. 6. Solubility of griseofulvin 11 in aqueous solutions withsulfoxide 3 (Δ) and DMSO (+) as cosolvents. Each trial was run induplicate and each point represents the average of the duplicate trials.In the case sulfoxide 3, the pH varied from 6.3 (with no additive) to6.8 (with a 0.20 weight fraction additive). In DMSO, the pH varied from6.3 (with no additive) to 6.8 (with a 0.20 weight fraction additive).

FIG. 7. Compounds assayed for PKD1 inhibitory activity. The structureshave been reported previously.

FIG. 8. Plot of PKD1 activity with compound concentrations of 1 μM.Stock solutions were prepared in three different media (DMSO (▪), NMP(□), and 25% 3/NMP (▪)) at concentrations of 10 mM, and dilutions wereperformed using the same media. The % PKD1 activity is reported as themean, and error bars represent SEM (n=3). The % PKD1 activity wasdetermined as previously described.

FIG. 9. Plot of PKD1 activity with compound concentrations of 10 μM.Stock solutions were prepared in three different media (DMSO (▪), NMP(□) and 25% 3/NMP (▪)) at concentrations of 10 mM, and dilutions wereperformed using the same media. The % PKD1 activity is reported as themean, and error bars represent SEM (n=3). The % PKD1 activity wasdetermined as previously described.

FIG. 10. The data are from a screening experiment where 32D cl 3 cellswere irradiated to doses of 0, 1, 3, 5, or 7 Gy and placed in T25flasks. Either JP4-039 or MM350 were added at 10 μM and incubated in aCO₂ incubator at 37° C. At Day 3 or 5 after irradiation, the cells werecounted and the viability determined. In these experiments MMS350 iscomparable to JP4-039 in the number of cells and viability.

FIG. 11. MMS350 was dissolved in PBS and injected IP at 20 mg/kg. Themice were irradiated to 9.5 Gy total body irradiation and then injectedat 4 or 24 hr after irradiation.

FIG. 12. In vitro radioprotection and mitigation of bone marrow stromalcells. In vitro survival curves were performed using a bone marrowstromal cell line derived from C57BL/6NTac mice. Cells were incubated in100 μM MMS350 for one hour before irradiation or was added to the cells10 min after irradiation. Cells were irradiated to doses ranging from 0to 8 Gy, plated in 4 well tissue culture plates, incubated for 7 days at37° C. in a humidified CO₂ incubator, and stained with crystal violet.Colonies of greater than 50 cells were counted and analyzed using alinear quadratic model or single-hit, multi-target model. Cellsincubated in MMS350 before irradiation were more resistant toirradiation as seen by an increased shoulder on the survival curve(ñ=14.9±2.9 compared to 5.8±1.1 for control cells, p=0.0039). Cellsadministered MMS350 following irradiation also were more radioresistantas demonstrated by an increased D₀ (2.4±0.3 compared to 1.9±0.1 forcontrol cells, p=0.0444).

FIG. 13. In vivo mitigation of total body irradiation by MMS350. MMS350was dissolved in water at a concentration of 2 mg/ml. C57BL/6NTac mice(n=15/group) were irradiated to 9.5 Gy total body irradiation andinjected intraperitoneally with 10 mg/kg of MMS350 at 4 or 24 hr. afterirradiation. The mice were followed for the development of thehematopoietic syndrome at which time the mice were sacrificed. Miceadministered MMS350 at either 4 or 24 hrs. after irradiation hadsignificantly increased survival (p=0.0092 and 0.0097, respectively)compared to 10 mg/kg JP4-039 at 24 hours.

FIG. 14. Pulmonary migration of luc+ marrow cells to irradiated lungs ofluc+ chimeric mice. Groups of 30 C57BL/6Tac mice were given 10 Gy TBIthen 24 hr. later 1×10⁷ luc+ bone marrow cells I.V. At day 63, subgroupsreceived 18 Gy thoracic irradiation. Total body irradiated, luc+ bonemarrow chimeric mice demonstrated marrow cavity specific bioluminescence(day 28) and no specific pulmonary concentration of luc+ cells until 60days after thoracic irradiation (day 123 post luc+ cells). Mice wereserially imaged for luciferase, as described in the methods. Arepresentative mice is shown in FIG. 14.

FIG. 15. Pulmonary migration of luc+ stromal cells to lungs ofirradiated mice is reduced by MMS350. C57BL/6NTac mice were irradiatedto 20 Gy to the pulmonary cavity. The mice were shielded so that onlythe pulmonary cavity was irradiated. On day 88 after irradiation, halfof the mice were placed on 100 mM MMS350 in drinking water. Insubgroups, at day: 3, 50, or 100 after irradiation, mice were injectedintraperitoneally with 1×10⁶ cells of a clonal bone marrow stromal cellline derived from a luciferase+ transgenic mouse. In vivo imagingrevealed little or no migration of luc+ BM stromal cells to the lungs of20 Gy thoracic irradiated mice on days 3-15 post irradiation (A, D) ordays 60-75 post irradiation (B,E). By 129 days post 20 Gy thoracicirradiation, migration of luc+ BM stromal cells to the lungs wassignificant (F). Mice on MMS350 in drinking water displayed asignificant decrease in pulmonary migration of luc+ bone marrow stromalcells compared to control mice (C).

FIG. 16. In vivo serial imaging of luc+ bone marrow stromal cells post20 Gy to the right hind leg. In vivo imaging revealed no migration of1×10⁶ luc+ BM stromal cells on days 1-15 (A, D), days 52-65 (B, E), ordays 140-155 (C, F) post 20 Gy to the right hind leg.

FIG. 17. Cell division of luciferase positive-bone marrow stromal cellline migrating to lungs Dual stained lung sections demonstratesimultaneously stained luc+ and BrdU+ cells (yellow) (A); and same luc+positivity stained ______ cells (green) (B); and BrdU+ only stainedcells (red) (C).

FIG. 18. Irradiation induction of mRNA by RT-PCR for acute phasereacting latent period and late fibrotic period transcript genes.

-   -   A. Acute phase reactants    -   B. Endothelial specific genes    -   C. Late fibrosis associated genes including TGF-β, IGFbp7,        MnSOD, TNF-α, lysl-oxidase, and TLR4

FIG. 19. MicroRNA expression during acute phase, latent period, and lateradiation fibrotic phase in lungs of 20 Gy irradiated mice.

-   -   A. TLR4/miRNA151, and miRNA107    -   B. VEGF/miRNA126    -   C. TGF-β/miRNA155

FIG. 20. MMS350 decreases levels of irradiation induced endothelial andalveolar cell specific RNA levels. Endothelial and alveolar cells wereisolated from the lungs of irradiated control or MMS350 treated mice atdays 0, 29, 150, and 200 after 20 Gy thoracic irradiation. Subgroups ofmice had received MMS350 continuously in the drinking water beginning 7days before irradiation. RNA was isolated and RT-PCR was performed forgene expression for: A) oxidative stress induced promoters; B)endothelial and alveolar cell markers; C) fibrosis associated genes; andD) endothelial cell markers. Gene expression that was increased by lungirradiation in endothelial cells was decreased in mice treated withMMS350 in the drinking water compared to the effect on pulmonary genesassociated with irradiated alveolar cells.

FIG. 21. MMS350 reduces irradiation induced bromodomain protein RNAtranscript levels associated with the late pulmonary fibrotic phase.Mice irradiated to 20 Gy to pulmonary cavity were sacrificed at varioustimes after irradiation. mRNA was extracted from: A) total lungs; B)endothelial cells; or C) alveolar II cells. Expression of BD1, BD2, BD3,and BRDT was determined.

FIG. 22. Conditional medium for endothelial cells removed from 20 Gythoracic cavity irradiated mice does not stimulate luc+ stromal cellmobility.

FIG. 23. MMS350 in drinking water decreases fibrosis in the lungs ofthoracic irradiated (20 Gy) mice.

FIG. 24 depicts the synthesis scheme of an azetidine compound.

FIG. 25 depicts the synthesis scheme of additional oxetanesulfoxide-substituted compounds.

DETAILED DESCRIPTION Terminology

As used herein, the singular terms “a,” “an,” and “the” include pluralreferents unless context clearly indicates otherwise. Also, as usedherein, the term “comprises” means “includes.”

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present disclosure,suitable methods and materials are described below. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

To facilitate review of the various examples of this disclosure, thefollowing explanations of specific terms are provided:

The terms “alkyl”, “aryl” and other groups refer generally to bothunsubstituted and substituted groups unless specified to the contrary.

The term acyl refers to —C(O)R^(f) wherein R^(f) is defined below.

“Administration” as used herein is inclusive of administration byanother person to the subject or self-administration by the subject.

The term “alkenyl” refers to a straight or branched chain hydrocarbongroup with at least one double bond, preferably with 2-15 carbon atoms,and more preferably with 2-10 carbon atoms (for example, —CH═CHR^(g) or—CH₂CH═CHR^(g), wherein R^(g) is defined below).

The term “alkoxy” refers to —OR^(d), wherein R^(d) is an alkyl group.Suitable alkoxy groups include methoxy, ethoxy, n-propoxy, i-propoxy,n-butoxy, i-butoxy, sec-butoxy, tert-butoxy cyclopropoxy, cyclohexyloxy,and the like.

Unless otherwise specified, alkyl groups are hydrocarbon groups and, ina number of embodiments, are C₁-C₁₆ (that is, having 1 to 16 carbonatoms) or C₁-C₁₀ alkyl groups. Alkyl groups can be branched orunbranched, acyclic or cyclic. The above definition of an alkyl groupand other definitions apply also when the group is a substituent onanother group (for example, an alkyl group as a substituent of atrialkylsilyl group). For example, a lower alkyl or (C₁-C₆)alkyl can bemethyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl, pentyl,3-pentyl, or hexyl; (C₃-C₆)cycloalkyl can be cyclopropyl, cyclobutyl,cyclopentyl, or cyclohexyl; (C₃-C₆)cycloalkyl(C₁-C₆)alkyl can becyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl,cyclohexylmethyl, 2-cyclopropylethyl, 2-cyclobutylethyl,2-cyclopentylethyl, or 2-cyclohexylethyl; (C₁-C₆)alkoxy can be methoxy,ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy,3-pentoxy, or hexyloxy; (C₂-C₆)alkenyl can be vinyl, allyl, 1-propenyl,2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1,-pentenyl, 2-pentenyl,3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, or5-hexenyl; (C₂-C₆)alkynyl can be ethynyl, 1-propynyl, 2-propynyl,1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl,4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, or 5-hexynyl;(C₁-C₆)alkanoyl can be acetyl, propanoyl or butanoyl; halo(C₁-C₆)alkylcan be iodomethyl, bromomethyl, chloromethyl, fluoromethyl,trifluoromethyl, 2-chloroethyl, 2-fluoroethyl, 2,2,2-trifluoroethyl, orpentafluoroethyl; hydroxy(C₁-C₆)alkyl can be hydroxymethyl,1-hydroxyethyl, 2-hydroxyethyl, 1-hydroxypropyl, 2-hydroxypropyl,3-hydroxypropyl, 1-hydroxybutyl, 4-hydroxybutyl, 1-hydroxypentyl,5-hydroxypentyl, 1-hydroxyhexyl, or 6-hydroxyhexyl;(C₁-C₆)alkoxycarbonyl can be methoxycarbonyl, ethoxycarbonyl,propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, orhexyloxycarbonyl; (C₁-C₆)alkylthio can be methylthio, ethylthio,propylthio, isopropylthio, butylthio, isobutylthio, pentylthio, orhexylthio; (C₂-C₆)alkanoyloxy can be acetoxy, propanoyloxy, butanoyloxy,isobutanoyloxy, pentanoyloxy, or hexanoyloxy.

The term “alkynyl” refers to a straight or branched chain hydrocarbongroup with at least one triple bond, preferably with 2-15 carbon atoms,and more preferably with 2-10 carbon atoms (for example, —C≡CR^(h) or—CH₂—C≡CR^(h), wherein R^(h) is defined below).

An “animal” refers to living multi-cellular vertebrate organisms, acategory that includes, for example, mammals and birds. The term mammalincludes both human and non-human mammals. Similarly, the term “subject”includes both human and non-human subjects, including birds andnon-human mammals, such as non-human primates, companion animals (suchas dogs and cats), livestock (such as pigs, sheep, cows), as well asnon-domesticated animals, such as the big cats. The term subject appliesregardless of the stage in the organism's life-cycle. Thus, the termsubject applies to an organism in utero or in ovo, depending on theorganism (that is, whether the organism is a mammal or a bird, such as adomesticated or wild fowl).

The term “aryl” refers to phenyl or naphthyl, or substituted phenyl ornaphthyl.

The term “aryloxy” refers to —OR^(e), wherein R^(e) is an aryl group.

The term “co-administration” or “co-administering” refers toadministration of a an oxetane-substituted compound with at least oneother therapeutic agent within the same general time period, and doesnot require administration at the same exact moment in time (althoughco-administration is inclusive of administering at the same exact momentin time). Thus, co-administration may be on the same day or on differentdays, or in the same week or in different weeks. The additionaltherapeutic agent may be included in the same composition as theoxetane-substituted compound.

The term “heteroaryl” refers to a monocyclic- or polycyclic aromaticring comprising carbon atoms, hydrogen atoms, and one or moreheteroatoms. In a number of embodiments, 1 to 3 heteroatoms are presentand are, independently selected from nitrogen, oxygen, phosphorus,sulfur, chlorine, bromine and iodine. As is known to those skilled inthe art, heteroaryl rings have less aromatic character than theirall-carbon counter parts. A heteroaryl group need thus only have somedegree of aromatic character. Illustrative examples of heteroaryl groupsinclude, but are not limited to, pyridinyl, pyridazinyl, pyrimidyl,pyrazyl, triazinyl, pyrrolyl, pyrazolyl, imidazolyl, (1,2,3,)- and(1,2,4)-triazolyl, pyrazinyl, pyrimidinyl, tetrazolyl, furyl, thienyl,isoxazolyl, thiazolyl, phenyl, isoxazolyl, and oxazolyl. A heteroarylgroup can be unsubstituted or substituted with, for example, one or twosubstituents. In a number of embodiments, heteroaryl groups hereininclude 2 to 11 carbon atoms (C₂-C₁₁ heteroaryl groups). In severalembodiments, heteroaryl group hereof are monocyclic rings, wherein thering comprises 2 to 10 carbon atoms or 3 to 6 carbon atoms and 1 to 3heteroatoms.

The groups set forth above, can be substituted with a wide variety ofsubstituents to synthesize analogs retaining desirable properties. Forexample, alkyl groups and other groups may be substituted with a groupor groups including, but not limited to, a halo group, a benzyl group, aphenyl group, an alkoxy group, a hydroxy group, an amino group(including, for example, free amino groups, alkylamino, dialkylaminogroups and arylamino groups), an alkenyl group, an alkynyl group and anacyloxy group. In the case of amino groups (—NR^(a)R^(b)), R^(a) andR^(b) are preferably independently hydrogen, an acyl group, an alkylgroup, or an aryl group. Acyl groups may, for example, be substitutedwith (that is, R^(f) is) an alkyl group, a haloalkyl group (for example,a perfluoroalkyl group), an alkoxy group, an amino group and a hydroxygroup. Alkynyl groups and alkenyl groups may preferably be substitutedwith (that is, R^(g) and R^(h) are, for example) a group or groupsincluding, but not limited to, an alkyl group, an alkoxyalkyl group, anamino alkyl group and a benzyl group.

“Inhibiting” refers to inhibiting the full development of a disease orcondition. “Inhibiting” also refers to any quantitative or qualitativereduction in biological activity or binding, relative to a control.

A “therapeutically effective amount” refers to a quantity of a specifiedagent sufficient to achieve a desired effect in a subject being treatedwith that agent. For example, a therapeutically amount may be an amountof an oxetane-substituted compound that is sufficient to inhibitradiation-induced damage in a subject. Ideally, a therapeuticallyeffective amount of an agent is an amount sufficient to inhibit or treatthe disease or condition without causing a substantial cytotoxic effectin the subject. The therapeutically effective amount of an agent will bedependent on the subject being treated, the severity of the affliction,and the manner of administration of the therapeutic composition.

“Treatment” refers to a therapeutic intervention that ameliorates a signor symptom of a disease or pathological condition after it has begun todevelop. As used herein, the term “ameliorating,” with reference to adisease or pathological condition, refers to any observable beneficialeffect of the treatment. The beneficial effect can be evidenced, forexample, by a delayed onset of clinical symptoms of the disease in asusceptible subject, a reduction in severity of some or all clinicalsymptoms of the disease, a slower progression of the disease, animprovement in the overall health or well-being of the subject, or byother parameters well known in the art that are specific to theparticular disease. The phrase “treating a disease” refers to inhibitingthe full development of a disease, for example, in a subject who is atrisk for a disease such as cancer. “Preventing” a disease or conditionrefers to prophylactic administering a composition to a subject who doesnot exhibit signs of a disease or exhibits only early signs for thepurpose of decreasing the risk of developing a pathology or condition,or diminishing the severity of a pathology or condition. In certainembodiments disclosed herein, the treatment inhibits radiation damage ina subject.

“Pharmaceutical compositions” are compositions that include an amount(for example, a unit dosage) of one or more of the disclosed compoundstogether with one or more non-toxic pharmaceutically acceptableadditives, including carriers, diluents, and/or adjuvants, andoptionally other biologically active ingredients. Such pharmaceuticalcompositions can be prepared by standard pharmaceutical formulationtechniques such as those disclosed in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa. (19th Edition).

The terms “pharmaceutically acceptable salt or ester” refers to salts oresters prepared by conventional means that include salts, e.g., ofinorganic and organic acids, including but not limited to hydrochloricacid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonicacid, ethanesulfonic acid, malic acid, acetic acid, oxalic acid,tartaric acid, citric acid, lactic acid, fumaric acid, succinic acid,maleic acid, salicylic acid, benzoic acid, phenylacetic acid, mandelicacid and the like. “Pharmaceutically acceptable salts” of the presentlydisclosed compounds also include those formed from cations such assodium, potassium, aluminum, calcium, lithium, magnesium, zinc, and frombases such as ammonia, ethylenediamine, N-methyl-glutamine, lysine,arginine, ornithine, choline, N,N′-dibenzylethylenediamine,chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine,diethylamine, piperazine, tris(hydroxymethyl)aminomethane, andtetramethylammonium hydroxide. These salts may be prepared by standardprocedures, for example by reacting the free acid with a suitableorganic or inorganic base. Any chemical compound recited in thisspecification may alternatively be administered as a pharmaceuticallyacceptable salt thereof. “Pharmaceutically acceptable salts” are alsoinclusive of the free acid, base, and zwitterionic forms. Descriptionsof suitable pharmaceutically acceptable salts can be found in Handbookof Pharmaceutical Salts, Properties, Selection and Use, Wiley VCH(2002). When compounds disclosed herein include an acidic function suchas a carboxy group, then suitable pharmaceutically acceptable cationpairs for the carboxy group are well known to those skilled in the artand include alkaline, alkaline earth, ammonium, quaternary ammoniumcations and the like. Such salts are known to those of skill in the art.For additional examples of “pharmacologically acceptable salts,” seeBerge et al., J. Pharm. Sci. 66:1 (1977).

“Pharmaceutically acceptable esters” includes those derived fromcompounds described herein that are modified to include a carboxylgroup. An in vivo hydrolysable ester is an ester, which is hydrolysed inthe human or animal body to produce the parent acid or alcohol.Representative esters thus include carboxylic acid esters in which thenon-carbonyl moiety of the carboxylic acid portion of the ester groupingis selected from straight or branched chain alkyl (for example, methyl,n-propyl, t-butyl, or n-butyl), cycloalkyl, alkoxyalkyl (for example,methoxymethyl), aralkyl (for example benzyl), aryloxyalkyl (for example,phenoxymethyl), aryl (for example, phenyl, optionally substituted by,for example, halogen, C₁₋₄ alkyl, or C₁₋₄ alkoxy) or amino); sulphonateesters, such as alkyl- or aralkylsulphonyl (for example,methanesulphonyl); or amino acid esters (for example, L-valyl orL-isoleucyl). A “pharmaceutically acceptable ester” also includesinorganic esters such as mono-, di-, or tri-phosphate esters. In suchesters, unless otherwise specified, any alkyl moiety presentadvantageously contains from 1 to 18 carbon atoms, particularly from 1to 6 carbon atoms, more particularly from 1 to 4 carbon atoms. Anycycloalkyl moiety present in such esters advantageously contains from 3to 6 carbon atoms. Any aryl moiety present in such esters advantageouslycomprises a phenyl group, optionally substituted as shown in thedefinition of carbocycylyl above. Pharmaceutically acceptable estersthus include C₁-C₂₂ fatty acid esters, such as acetyl, t-butyl or longchain straight or branched unsaturated or omega-6 monounsaturated fattyacids such as palmoyl, stearoyl and the like. Alternative aryl orheteroaryl esters include benzoyl, pyridylmethyloyl and the like any ofwhich may be substituted, as defined in carbocyclyl above. Additionalpharmaceutically acceptable esters include aliphatic L-amino acid esterssuch as leucyl, isoleucyl and especially valyl.

For therapeutic use, salts of the compounds are those wherein thecounter-ion is pharmaceutically acceptable. However, salts of acids andbases which are non-pharmaceutically acceptable may also find use, forexample, in the preparation or purification of a pharmaceuticallyacceptable compound.

The pharmaceutically acceptable acid and base addition salts asmentioned hereinabove are meant to comprise the therapeutically activenon-toxic acid and base addition salt forms which the compounds are ableto form. The pharmaceutically acceptable acid addition salts canconveniently be obtained by treating the base form with such appropriateacid. Appropriate acids comprise, for example, inorganic acids such ashydrohalic acids, e.g. hydrochloric or hydrobromic acid, sulfuric,nitric, phosphoric and the like acids; or organic acids such as, forexample, acetic, propanoic, hydroxyacetic, lactic, pyruvic, oxalic (i.e.ethanedioic), malonic, succinic (i.e. butanedioic acid), maleic,fumaric, malic (i.e. hydroxybutanedioic acid), tartaric, citric,methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic,cyclamic, salicylic, p-aminosalicylic, pamoic and the like acids.Conversely said salt forms can be converted by treatment with anappropriate base into the free base form.

The compounds containing an acidic proton may also be converted intotheir non-toxic metal or amine addition salt forms by treatment withappropriate organic and inorganic bases. Appropriate base salt formscomprise, for example, the ammonium salts, the alkali and earth alkalinemetal salts, e.g. the lithium, sodium, potassium, magnesium, calciumsalts and the like, salts with organic bases, e.g. the benzathine,N-methyl-D-glucamine, hydrabamine salts, and salts with amino acids suchas, for example, arginine, lysine and the like.

The term “addition salt” as used hereinabove also comprises the solvateswhich the compounds described herein are able to form. Such solvates arefor example hydrates, alcoholates and the like.

The term “quaternary amine” as used hereinbefore defines the quaternaryammonium salts which the compounds are able to form by reaction betweena basic nitrogen of a compound and an appropriate quaternizing agent,such as, for example, an optionally substituted alkylhalide, arylhalideor arylalkylhalide, e.g. methyliodide or benzyliodide. Other reactantswith good leaving groups may also be used, such as alkyltrifluoromethanesulfonates, alkyl methanesulfonates, and alkylp-toluenesulfonates. A quaternary amine has a positively chargednitrogen. Pharmaceutically acceptable counterions include chloro, bromo,iodo, trifluoroacetate and acetate. The counterion of choice can beintroduced using ion exchange resins.

Some of the compounds described herein may also exist in theirtautomeric form.

Prodrugs of the disclosed compounds also are contemplated herein. Aprodrug is an active or inactive compound that is modified chemicallythrough in vivo physiological action, such as hydrolysis, metabolism andthe like, into an active compound following administration of theprodrug to a subject. The term “prodrug” as used throughout this textmeans the pharmacologically acceptable derivatives such as esters,amides and phosphates, such that the resulting in vivo biotransformationproduct of the derivative is the active drug as defined in the compoundsdescribed herein. Prodrugs preferably have excellent aqueous solubility,increased bioavailability and are readily metabolized into the activeinhibitors in vivo. Prodrugs of a compounds described herein may beprepared by modifying functional groups present in the compound in sucha way that the modifications are cleaved, either by routine manipulationor in vivo, to the parent compound. The suitability and techniquesinvolved in making and using prodrugs are well known by those skilled inthe art. F or a general discussion of prodrugs involving esters seeSvensson and Tunek, Drug Metabolism Reviews 165 (1988) and Bundgaard,Design of Prodrugs, Elsevier (1985).

The term “prodrug” also is intended to include any covalently bondedcarriers that release an active parent drug of the present invention invivo when the prodrug is administered to a subject. Since prodrugs oftenhave enhanced properties relative to the active agent pharmaceutical,such as, solubility and bioavailability, the compounds disclosed hereincan be delivered in prodrug form. Thus, also contemplated are prodrugsof the presently disclosed compounds, methods of delivering prodrugs andcompositions containing such prodrugs. Prodrugs of the disclosedcompounds typically are prepared by modifying one or more functionalgroups present in the compound in such a way that the modifications arecleaved, either in routine manipulation or in vivo, to yield the parentcompound. Prodrugs include compounds having a phosphonate and/or aminogroup functionalized with any group that is cleaved in vivo to yield thecorresponding amino and/or phosphonate group, respectively. Examples ofprodrugs include, without limitation, compounds having an acylated aminogroup and/or a phosphonate ester or phosphonate amide group. Inparticular examples, a prodrug is a lower alkyl phosphonate ester, suchas an isopropyl phosphonate ester.

Protected derivatives of the disclosed compounds also are contemplated.A variety of suitable protecting groups for use with the disclosedcompounds are disclosed in Greene and Wuts, Protective Groups in OrganicSynthesis; 3rd Ed.; John Wiley & Sons, New York, 1999.

In general, protecting groups are removed under conditions that will notaffect the remaining portion of the molecule. These methods are wellknown in the art and include acid hydrolysis, hydrogenolysis and thelike. One preferred method involves the removal of an ester, such ascleavage of a phosphonate ester using Lewis acidic conditions, such asin TMS-Br mediated ester cleavage to yield the free phosphonate. Asecond preferred method involves removal of a protecting group, such asremoval of a benzyl group by hydrogenolysis utilizing palladium oncarbon in a suitable solvent system such as an alcohol, acetic acid, andthe like or mixtures thereof. A t-butoxy-based group, including t-butoxycarbonyl protecting groups can be removed utilizing an inorganic ororganic acid, such as HCl or trifluoroacetic acid, in a suitable solventsystem, such as water, dioxane and/or methylene chloride. Anotherexemplary protecting group, suitable for protecting amino and hydroxyfunctions amino is trityl. Other conventional protecting groups areknown and suitable protecting groups can be selected by those of skillin the art in consultation with Greene and Wuts, Protective Groups inOrganic Synthesis; 3rd Ed.; John Wiley & Sons, New York, 1999. When anamine is deprotected, the resulting salt can readily be neutralized toyield the free amine. Similarly, when an acid moiety, such as aphosphonic acid moiety is unveiled, the compound may be isolated as theacid compound or as a salt thereof.

Particular examples of the presently disclosed compounds include one ormore asymmetric centers; thus these compounds can exist in differentstereoisomeric forms. Accordingly, compounds and compositions may beprovided as individual pure enantiomers or as stereoisomeric mixtures,including racemic mixtures. In certain embodiments the compoundsdisclosed herein are synthesized in or are purified to be insubstantially enantiopure form, such as in a 90% enantiomeric excess, a95% enantiomeric excess, a 97% enantiomeric excess or even in greaterthan a 99% enantiomeric excess, such as in enantiopure form.

Overview

Oxetane-substituted compounds, including novel oxetane-substitutedsulfoxide, sulfide and sulfone compounds, hereof have demonstratedradiation protection activities. Oxetane-substituted compounds hereofmay, for example, effect or provide radioprotection and/orradiomitigation, for example, upon applying or administering aneffective dosage thereof. It is anticipated that effective dosages willbe in the range of approximately 1 mg/kg to 150 mg/kg. As known to thoseskilled in the art, however, effective dosage sizes will vary accordingto the route of administration, and the frequency of administration.

The compounds disclosed herein are useful as radiomitigators to prevent,mitigate, or treat radiation induced damage to cells tissues, or organs,and/or organisms, that have already been exposed to radiation (e.g.,from clinical or non-clinical sources), or as radioprotectors tomitigate or prevent damage to cells tissues or organs, and/or organismsthat are expected to be exposed to radiation (e.g., in anticipation ofradiotherapy, in certain military contexts, and the like).

For example, a water soluble oxetanyl sulfoxide (referred to herein as“MMS350′”) was evaluated as a radiation protector and mitigator. MMS350was effective both as a protector and mitigator of clonal mouse bonemarrow stromal cell lines in vitro. Single dose administration of MMS35024 hours after 9.5 Gy total body irradiation of C57BL/6/HNsd miceresulted in significant improvement in survival. Thoracic irradiatedmice (20 Gy) demonstrated acute mRNA elevations for: TGF-β, IL-1, TNF-α,MnsOD, NFK-B, Nrf2, SP1, AP1, and TLR4. During the latent period (days14-100), endothelial cell localized elevation of vWF, VEGF, CCL3, CTGF,and IL6 was detected followed by MnSOD and TGF-β after day 100. MMS350(100 mM) added daily to drinking water beginning at day 88 after 20 Gythoracic irradiation substantially decreased pulmonary inflammatory andpro-fibrotic gene expression, migration into the lungs of bone marroworigin luciferase+/GFP+ fibroblast progenitors in (both marrow chimericand luciferase+ (luc+/GFP) stromal cell line injected mice), andradiation fibrosis (p<0.0001). MMS350 decreased radiation pulmonaryfibrosis in both marrow chimeric and luc+ stromal cell line injectedmice, and significantly increased survival. In summary, MMS350 whendelivered over several weeks (e.g. at least 2 weeks or 2-5 weeks) indrinking water reduces late irradiation induced biomarker elevation andmarrow stromal cell mediated pulmonary fibrosis in C57BL/6NTac mice. Thenon-toxic and orally bioavailable small molecule radiation mitigatorsdisclosed herein should prove an effective counter measure against bothacute and chronic effects of ionizing irradiation.

Oxetane-substituted compounds hereof may also have potential as, forexample, a DMSO substitute for enhancing the dissolution of organiccompounds with poor aqueous solubilities. Such compounds may, forexample, provide utility in applications of library storage andbiological assays (as, for example, a DMSO substitute).

For example, the use of MMS350 3 (FIG. 1) as a solubilizer and generalcompound storage additive was evaluated. It was found that addition ofsulfoxide 3 increased the aqueous solubility of several model “problem”compounds including naproxen, quinine, curcumin, carbendazim, andgriseofulvin. The solubility enhancement surpassed that of DMSO at massfractions greater than 10%.

Compounds

In a number of embodiments, oxetane substituted compounds hereof havethe formula I:

wherein X is S, SO or SO₂;

one of R¹, R², and R³ is O and the others of R¹, R² and R³ areindependently, the same or different, CH₂, or CR¹³ wherein, R¹³ is analkyl group, an alkenyl group, an alkynyl group, a trialkylsilyl group,or —(CH₂)_(m)OR¹⁵, wherein R¹⁵ is an alkyl group or an aryl group and mis an integer in the range of 1 to 10, one of R⁵, R⁶, and R⁷ is O andthe others of R⁵, R⁶ and R⁷ are independently, the same or different,CH₂, or CR¹⁴ wherein, R¹⁴ is an alkyl group, an alkenyl group, analkynyl group, a trialkylsilyl group, or —(CH₂)_(n)OR¹⁶, wherein R¹⁶ isan alkyl group or an aryl group and n is an integer in the range of 1 to10;

R⁴ and R⁸ are independently, the same or different, H, an alkyl group,an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, aC₁-C₃ alkoxy group, an aryloxy group, or —(CH₂)_(q)OR¹⁷, wherein R¹⁷ isan alkyl group or an aryl group and q is an integer in the range of 1 to10, provided that R⁴ is not a C₁-C₃ alkoxy group or an aryloxy groupwhen R¹ or R³ is O and R⁸ is not a C₁-C₃ alkoxy group or an aryloxygroup when R⁵ or R⁷ is O;

R⁹, R¹⁰, R¹¹ and R¹² are independently, the same or different, H, analkyl group, an alkenyl group, an alkynyl group, an aryl group.

In a number of embodiments, R¹³ is a C₁-C₃ alkyl group, a C₂-C₃ alkenylgroup, a C₂-C₃ alkynyl group, or a trialkylsilyl group and R¹⁴ is aC₁-C₃ alkyl group, a C₂-C₃ alkenyl group, a C₂-C₃ alkynyl group, or atrialkylsilyl group.

In a number of embodiments, one of R⁹ and R¹⁰ is H and one of R¹¹ andR¹² is H.

In a number of embodiments, one of R¹, R², and R³ is O and the others ofR¹, R² and R³ are CH₂, and one of R⁵, R⁶, and R⁷ is O and the others ofR⁵, R⁶ and R⁷ are CH₂.

In certain embodiments, R² and R⁶ are each O; and R¹, R³, R⁵, R⁷ areeach CH₂.

In certain embodiments, R² and R⁶ are each O; R¹, R³, R⁵, R⁷ are eachCH₂; R⁴ and R⁸ are each C₁-C₁₀ alkyl such as a methyl, ethyl, propyl,isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, or hexyl; andR⁹-R¹² are each H.

In certain embodiments, R⁹-R¹² are each H.

In certain embodiments, X is SO; R² and R⁶ are each O; R¹, R³, R⁵, R⁷are each CH₂; R⁴ and R⁸ are each C₁-C₁₀ alkyl such as a methyl, ethyl,propyl, isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, orhexyl; and R⁹-R¹² are each H.

In other embodiments of formula I, one of R¹, R², and R³ is NR⁶⁰ and theothers of R¹, R² and R³ are independently, the same or different, CH₂,or CR¹³ wherein, R¹³ is an alkyl group, an alkenyl group, an alkynylgroup, a trialkylsilyl group, or —(CH₂)_(m)OR¹⁵, wherein R¹⁵ is an alkylgroup or an aryl group and m is an integer in the range of 1 to 10; oneof R⁵, R⁶, and R⁷ is NR⁶¹ and the others of R⁵, R⁶ and R⁷ areindependently, the same or different, CH₂, or CR¹⁴ wherein, R¹⁴ is analkyl group, an alkenyl group, an alkynyl group, a trialkylsilyl group,or —(CH₂)_(n)OR¹⁶, wherein R¹⁶ is an alkyl group or an aryl group and nis an integer in the range of 1 to 10, wherein R⁶⁰ and R⁶¹ are eachindependently H, an alkyl group, an alkenyl group, an alkynyl group, anaryl group or a heteroaryl group; and X, R⁴, and R⁸-R¹² are the same asabove. In certain embodiments, R⁶⁰ and R⁶¹ are each independently anaryl group substituted with an alkoxy group (e.g., a lower alkoxy group)or a halo group, particularly para-substituted. FIG. 19 depicts asynthetic scheme for synthesizing these azetidine-containing compounds.

Also disclosed herein are pharmaceutically acceptable salts or esters ofthe oxetane-substituted compounds.

In several representative embodiments, the compound has the formula

In a number of representative radiation studies, the following compoundwas studied:

(referred to herein as “MMS350”)

As discussed above, the present inventors have also discovered thatoxetane-substituted compounds can be used for enhancing the solubilityof organic compounds in aqueous media. For model or representativecompounds studied, significant solubility enhancements were observedusing a representative oxetane-substituted sulfoxide as a cosolvent inaqueous media. In a number of studies, the representativeoxetane-substituted sulfoxide had the formula:

Brine shrimp, breast cancer (MDA-MB-231) and liver cell line (HepG2)toxicity data for the above sulfoxide are set forth below, in additionto comparative IC50 values for a series of PKD1 inhibitors. Radiationdamage mitigation studies of the above sulfoxide are set forth below.

In certain embodiments, the oxetane-substituted compounds are solids atroom temperature and pressure. In certain embodiments, theoxetane-substituted compounds are water soluble or water miscible andthus may be mixed with water to form an aqueous solution or medium. Infurther embodiments, the oxetane-substituted compounds may be mixed withan appropriate water-soluble cosolvent to form an aqueous solution.Illustrative cosolvents include N-methyl-2-pyrrolidone (NMP), dimethylsulfoxide (DMSO), alcoholic solvents (e.g., ethanol or isopropylalcohol), and acetonitrile.

Also disclosed herein are compounds, and pharmaceutically acceptablesalts and esters, thereof, having a formula II:

wherein X is S, SO or SO₂;

one of R¹, R², and R³ is O and the others of R¹, R² and R³ areindependently, the same or different, CH₂, or CR¹³ wherein, R¹³ is analkyl group, an alkenyl group, an alkynyl group, a trialkylsilyl group,or —(CH₂)_(m)OR¹⁵, wherein R¹⁵ is an alkyl group or an aryl group and mis an integer in the range of 1 to 10;

R⁴ is H, an alkyl group, an alkenyl group, an alkynyl group, an arylgroup, a heteroaryl group, a C₁-C₃ alkoxy group, an aryloxy group, or—(CH₂)_(q)OR¹⁷, wherein R¹⁷ is an alkyl group or an aryl group and q isan integer in the range of 1 to 10;

R⁹, R¹⁰, R¹¹ and R¹² are independently, the same or different, H, analkyl group, an alkenyl group, an alkynyl group, an aryl group.

In certain embodiments of formula II, R² is O; and R¹ and R³ are eachCH₂.

In certain embodiments of formula II, R² is O; R¹ and R³ are each CH₂;R⁴ is C₁-C₁₀ alkyl such as a methyl, ethyl, propyl, isopropyl, butyl,iso-butyl, sec-butyl, pentyl, 3-pentyl, or hexyl; and R⁹-R¹² are each H.

In certain embodiments of formula II, R⁹-R¹² are each H.

In certain embodiments of formula II, X is SO; is O; R¹ and R³ are eachCH₂; R⁴ is C₁-C₁₀ alkyl such as a methyl, ethyl, propyl, isopropyl,butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, or hexyl; and R⁹-R¹² areeach H.

Also disclosed herein are compounds, and pharmaceutically acceptablesalts and esters thereof, having a formula III:

wherein R³³ includes an oxetanyl sulfane, oxetanyl sulfinyl, or oxetanylsulfonyl moiety;

X is one of

R³¹, R³² and R³⁴ are, independently, hydrogen, C₁-C₆ straight orbranched-chain alkyl, optionally including a phenyl (C₆H₅) group, thatoptionally is methyl-, hydroxyl- or fluoro-substituted, including:methyl, ethyl, propyl, 2-propyl, butyl, t-butyl, pentyl, hexyl, benzyl,hydroxybenzyl (e.g., 4-hydroxybenzyl), phenyl and hydroxyphenyl. R³⁰ is—NH—R³⁵, —O—R³⁵ or —CH₂—R³⁵, where R³⁵ is an —N—O., —N—OH or N═Ocontaining group. In one embodiment, R³⁰ is

(1-Me-AZADO or 1-methyl azaadamantane N-oxyl). In another embodiment,R³⁰ is

(TMIO; 1,1,3,3-tetramethylisoindolin-2-yloxyl). In a further embodiment,R³⁰ is

In certain embodiments, R³³ may be —C(O)—(CH₂)_(x)—S—CH₂-oxetanyl-CH₃,—C(O)—(CH₂)_(x)—SO—CH₂-oxetanyl-CH₃,—C(O)—(CH₂)_(x)—SO₂—CH₂-oxetanyl-CH₃,—C(O)O—(CH₂)_(x)—S—CH₂-oxetanyl-CH₃,—C(O)O—(CH₂)_(x)—SO—CH₂-oxetanyl-CH₃, or—C(O)O—(CH₂)_(x)—SO₂—CH₂-oxetanyl-CH₃, wherein x is selected from 0 to5, more particularly 1 to 3, and most particularly 2.

In certain embodiments of formula III, R³³ is—C(O)O—(CH₂)_(x)—SO—CH₂-oxetanyl-CH₃; R³¹, R³² and R³⁴ are eachindependently hydrogen, C₁-C₆ straight or branched-chain alkyl, orphenyl-substituted C₁-C₆ straight or branched-chain alkyl; and R³⁰ is

An example of a compound of formula III is compound 9 in FIG. 25.

Also disclosed herein are compounds, and pharmaceutically acceptablesalts and esters thereof, having a formula IV:

or a formula V

wherein R⁴⁰ includes an oxetanyl sulfane, oxetanyl sulfinyl, or oxetanylsulfonyl moiety;R⁴¹, R^(41a), R⁴⁴, and R^(44a) are each independently hydrogen, a halo,C₁-C₆ straight or branched-chain alkyl, or a C₁-C₆ straight orbranched-chain alkyl further comprising a phenyl (C₆H₅) group, whereinthe C₁-C₆ straight or branched-chain alkyl group or the C₁-C₆ straightor branched-chain alkyl group comprising a phenyl group is unsubstitutedor is methyl-, hydroxyl- or halo-substituted; R₄ is hydrogen, a halo, aC₁-C₆ straight or branched-chain alkyl, or a C₁-C₆ straight orbranched-chain alkyl further comprising a phenyl (C₆H₅) group, whereinthe C₁-C₆ straight or branched-chain alkyl group or the C₁-C₆ straightor branched-chain alkyl group comprising a phenyl group is unsubstitutedor is methyl-, hydroxyl- or halo-substituted;R⁴⁵ is an —N—O., —N—OH or N═O containing group;R⁴², R⁴³, R⁴⁶, and R^(46a) are independently H or a halo, a C₁-C₆straight or branched-chain alkyl, or a C₁-C₆ straight or branched-chainalkyl further comprising a phenyl (C₆H₅) group, wherein the C₁-C₆straight or branched-chain alkyl group or the C₁-C₆ straight orbranched-chain alkyl group comprising a phenyl group is unsubstituted oris methyl-, hydroxyl- or halo-substituted.In certain embodiments, R⁴⁰ may be —C(O)—(CH₂)_(x)—S—CH₂-oxetanyl-CH₃,—C(O)—(CH₂)_(x)—SO—CH₂-oxetanyl-CH₃,—C(O)—(CH₂)_(x)—SO₂—CH₂-oxetanyl-CH₃,—C(O)O—(CH₂)_(x)—S—CH₂-oxetanyl-CH₃,—C(O)O—(CH₂)_(x)—SO—CH₂-oxetanyl-CH₃, or—C(O)O—(CH₂)_(x)—SO₂—CH₂-oxetanyl-CH₃, wherein x is selected from 0 to5, more particularly 1 to 3, and most particularly 2.Two examples of a compound of formula IV are shown below:

Also disclosed herein are compounds, and pharmaceutically acceptablesalts and esters thereof, having a formula VI:

wherein R²² includes an oxetanyl sulfane, oxetanyl sulfinyl, or oxetanylsulfonyl moiety;R²³, R^(23a), R²¹, and R^(21a) are each independently hydrogen, a halo,C₁-C₆ straight or branched-chain alkyl, or a C₁-C₆ straight orbranched-chain alkyl further comprising a phenyl (C₆H₅) group, whereinthe C₁-C₆ straight or branched-chain alkyl group or the C₁-C₆ straightor branched-chain alkyl group comprising a phenyl group is unsubstitutedor is methyl-, hydroxyl- or halo-substituted; R₄ is hydrogen, a halo, aC₁-C₆ straight or branched-chain alkyl, or a C₁-C₆ straight orbranched-chain alkyl further comprising a phenyl (C₆H₅) group, whereinthe C₁-C₆ straight or branched-chain alkyl group or the C₁-C₆ straightor branched-chain alkyl group comprising a phenyl group is unsubstitutedor is methyl-, hydroxyl- or halo-substituted; andR²⁰ is an —N—O., —N—OH or N═O containing group.In certain embodiments, R²² may be —C(O)—(CH₂)_(x)—S—CH₂-oxetanyl-CH₃,—C(O)—(CH₂)_(x)—SO—CH₂-oxetanyl-CH₃,—C(O)—(CH₂)_(x)—SO₂—CH₂-oxetanyl-CH₃,—C(O)O—(CH₂)_(x)—S—CH₂-oxetanyl-CH₃,—C(O)O—(CH₂)_(x)—SO—CH₂-oxetanyl-CH₃, or—C(O)O—(CH₂)_(x)—SO₂—CH₂-oxetanyl-CH₃, wherein x is selected from 0 to5, more particularly 1 to 3, and most particularly 2.An example of a compound of formula VI is compound II in FIG. 25.

Compositions

Another aspect of the disclosure includes pharmaceutical compositionsprepared for administration to a subject and which include atherapeutically effective amount of one or more of the compoundsdisclosed herein. The therapeutically effective amount of a disclosedcompound will depend on the route of administration, the species ofsubject and the physical characteristics of the subject being treated.Specific factors that can be taken into account include disease severityand stage, weight, diet and concurrent medications. The relationship ofthese factors to determining a therapeutically effective amount of thedisclosed compounds is understood by those of skill in the art.

Pharmaceutical compositions for administration to a subject can includeat least one further pharmaceutically acceptable additive such ascarriers, thickeners, diluents, buffers, preservatives, surface activeagents and the like in addition to the molecule of choice.Pharmaceutical compositions can also include one or more additionalactive ingredients such as antimicrobial agents, anti-inflammatoryagents, anesthetics, and the like. The pharmaceutically acceptablecarriers useful for these formulations are conventional. Remington'sPharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton,Pa., 19th Edition (1995), describes compositions and formulationssuitable for pharmaceutical delivery of the compounds herein disclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually contain injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (for example, powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. In addition to biologically-neutral carriers,pharmaceutical compositions to be administered can contain minor amountsof non-toxic auxiliary substances, such as wetting or emulsifyingagents, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate.

Pharmaceutical compositions disclosed herein include those formed frompharmaceutically acceptable salts and/or solvates of the disclosedcompounds. Pharmaceutically acceptable salts include those derived frompharmaceutically acceptable inorganic or organic bases and acids.Particular disclosed compounds possess at least one basic group that canform acid-base salts with acids. Examples of basic groups include, butare not limited to, amino and imino groups. Examples of inorganic acidsthat can form salts with such basic groups include, but are not limitedto, mineral acids such as hydrochloric acid, hydrobromic acid, sulfuricacid or phosphoric acid. Basic groups also can form salts with organiccarboxylic acids, sulfonic acids, sulfo acids or phospho acids orN-substituted sulfamic acid, for example acetic acid, propionic acid,glycolic acid, succinic acid, maleic acid, hydroxymaleic acid,methylmaleic acid, fumaric acid, malic acid, tartaric acid, gluconicacid, glucaric acid, glucuronic acid, citric acid, benzoic acid,cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic acid,2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid, nicotinicacid or isonicotinic acid, and, in addition, with amino acids, forexample with α-amino acids, and also with methanesulfonic acid,ethanesulfonic acid, 2-hydroxymethanesulfonic acid,ethane-1,2-disulfonic acid, benzenedisulfonic acid,4-methylbenzenesulfonic acid, naphthalene-2-sulfonic acid, 2- or3-phosphoglycerate, glucose-6-phosphate or N-cyclohexylsulfamic acid(with formation of the cyclamates) or with other acidic organiccompounds, such as ascorbic acid. In particular, suitable salts includethose derived from alkali metals such as potassium and sodium, alkalineearth metals such as calcium and magnesium, among numerous other acidswell known in the pharmaceutical art.

Certain compounds include at least one acidic group that can form anacid-base salt with an inorganic or organic base. Examples of saltsformed from inorganic bases include salts of the presently disclosedcompounds with alkali metals such as potassium and sodium, alkalineearth metals, including calcium and magnesium and the like. Similarly,salts of acidic compounds with an organic base, such as an amine (asused herein terms that refer to amines should be understood to includetheir conjugate acids unless the context clearly indicates that the freeamine is intended) are contemplated, including salts formed with basicamino acids, aliphatic amines, heterocyclic amines, aromatic amines,pyridines, guanidines and amidines. Of the aliphatic amines, the acyclicaliphatic amines, and cyclic and acyclic di- and tri-alkyl amines areparticularly suitable for use in the disclosed compounds. In addition,quaternary ammonium counterions also can be used.

Particular examples of suitable amine bases (and their correspondingammonium ions) for use in the present compounds include, withoutlimitation, pyridine, N,N-dimethylaminopyridine, diazabicyclononane,diazabicycloundecene, N-methyl-N-ethylamine, diethylamine,triethylamine, diisopropylethylamine, mono-, bis- ortris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine,tris(hydroxymethyl)methylamine, N,N-dimethyl-N-(2-hydroxyethyl)amine,tri-(2-hydroxyethyl)amine and N-methyl-D-glucamine. For additionalexamples of “pharmacologically acceptable salts,” see Berge et al., J.Pharm. Sci. 66:1 (1977).

Compounds disclosed herein can be crystallized and can be provided in asingle crystalline form or as a combination of different crystalpolymorphs. As such, the compounds can be provided in one or morephysical form, such as different crystal forms, crystalline, liquidcrystalline or non-crystalline (amorphous) forms. Such differentphysical forms of the compounds can be prepared using, for exampledifferent solvents or different mixtures of solvents forrecrystallization. Alternatively or additionally, different polymorphscan be prepared, for example, by performing recrystallizations atdifferent temperatures and/or by altering cooling rates duringrecrystallization. The presence of polymorphs can be determined by X-raycrystallography, or in some cases by another spectroscopic technique,such as solid phase NMR spectroscopy, IR spectroscopy, or bydifferential scanning calorimetry.

The pharmaceutical compositions can be administered to subjects by avariety of mucosal administration modes, including by oral, rectal,intranasal, intrapulmonary, or transdermal delivery, or by topicaldelivery to other surfaces. Optionally, the compositions can beadministered by non-mucosal routes, including by intramuscular,subcutaneous, intravenous, intra-arterial, intra-articular,intraperitoneal, intrathecal, intracerebroventricular, or parenteralroutes. In other alternative embodiments, the compound can beadministered ex vivo by direct exposure to cells, tissues or organsoriginating from a subject.

To formulate the pharmaceutical compositions, the compound can becombined with various pharmaceutically acceptable additives, as well asa base or vehicle for dispersion of the compound. Desired additivesinclude, but are not limited to, pH control agents, such as arginine,sodium hydroxide, glycine, hydrochloric acid, citric acid, and the like.In addition, local anesthetics (for example, benzyl alcohol),isotonizing agents (for example, sodium chloride, mannitol, sorbitol),adsorption inhibitors (for example, Tween 80 or Miglyol 812), solubilityenhancing agents (for example, cyclodextrins and derivatives thereof),stabilizers (for example, serum albumin), and reducing agents (forexample, glutathione) can be included. Adjuvants, such as aluminumhydroxide (for example, Amphogel, Wyeth Laboratories, Madison, N.J.),Freund's adjuvant, MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa,Hamilton, Ind.) and IL-12 (Genetics Institute, Cambridge, Mass.), amongmany other suitable adjuvants well known in the art, can be included inthe compositions. When the composition is a liquid, the tonicity of theformulation, as measured with reference to the tonicity of 0.9% (w/v)physiological saline solution taken as unity, is typically adjusted to avalue at which no substantial, irreversible tissue damage will beinduced at the site of administration. Generally, the tonicity of thesolution is adjusted to a value of about 0.3 to about 3.0, such as about0.5 to about 2.0, or about 0.8 to about 1.7.

The compound can be dispersed in a base or vehicle, which can include ahydrophilic compound having a capacity to disperse the compound, and anydesired additives. The base can be selected from a wide range ofsuitable compounds, including but not limited to, copolymers ofpolycarboxylic acids or salts thereof, carboxylic anhydrides (forexample, maleic anhydride) with other monomers (for example, methyl(meth)acrylate, acrylic acid and the like), hydrophilic vinyl polymers,such as polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone,cellulose derivatives, such as hydroxymethylcellulose,hydroxypropylcellulose and the like, and natural polymers, such aschitosan, collagen, sodium alginate, gelatin, hyaluronic acid, andnontoxic metal salts thereof. Often, a biodegradable polymer is selectedas a base or vehicle, for example, polylactic acid, poly(lacticacid-glycolic acid) copolymer, polyhydroxybutyric acid,poly(hydroxybutyric acid-glycolic acid) copolymer and mixtures thereof.Alternatively or additionally, synthetic fatty acid esters such aspolyglycerin fatty acid esters, sucrose fatty acid esters and the likecan be employed as vehicles. Hydrophilic polymers and other vehicles canbe used alone or in combination, and enhanced structural integrity canbe imparted to the vehicle by partial crystallization, ionic bonding,cross-linking and the like. The vehicle can be provided in a variety offorms, including fluid or viscous solutions, gels, pastes, powders,microspheres and films for direct application to a mucosal surface.

The compound can be combined with the base or vehicle according to avariety of methods, and release of the compound can be by diffusion,disintegration of the vehicle, or associated formation of waterchannels. In some circumstances, the compound is dispersed inmicrocapsules (microspheres) or nanocapsules (nanospheres) prepared froma suitable polymer, for example, isobutyl 2-cyanoacrylate (see, forexample, Michael et al., J. Pharmacy Pharmacol. 43:1-5, 1991), anddispersed in a biocompatible dispersing medium, which yields sustaineddelivery and biological activity over a protracted time.

The compositions of the disclosure can alternatively contain aspharmaceutically acceptable vehicles substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents and the like, forexample, sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate, and triethanolamineoleate. For solid compositions, conventional nontoxic pharmaceuticallyacceptable vehicles can be used which include, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharin, talcum, cellulose, glucose, sucrose, magnesiumcarbonate, and the like.

Pharmaceutical compositions for administering the compound can also beformulated as a solution, microemulsion, or other ordered structuresuitable for high concentration of active ingredients. The vehicle canbe a solvent or dispersion medium containing, for example, water,ethanol, polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycol, and the like), and suitable mixtures thereof.Proper fluidity for solutions can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of a desired particlesize in the case of dispersible formulations, and by the use ofsurfactants. In many cases, it will be desirable to include isotonicagents, for example, sugars, polyalcohols, such as mannitol andsorbitol, or sodium chloride in the composition. Prolonged absorption ofthe compound can be brought about by including in the composition anagent which delays absorption, for example, monostearate salts andgelatin.

In certain embodiments, the compound can be administered in a timerelease formulation, for example in a composition which includes a slowrelease polymer. These compositions can be prepared with vehicles thatwill protect against rapid release, for example a controlled releasevehicle such as a polymer, microencapsulated delivery system orbioadhesive gel. Prolonged delivery in various compositions of thedisclosure can be brought about by including in the composition agentsthat delay absorption, for example, aluminum monostearate hydrogels andgelatin. When controlled release formulations are desired, controlledrelease binders suitable for use in accordance with the disclosureinclude any biocompatible controlled release material which is inert tothe active agent and which is capable of incorporating the compoundand/or other biologically active agent. Numerous such materials areknown in the art. Useful controlled-release binders are materials thatare metabolized slowly under physiological conditions following theirdelivery (for example, at a mucosal surface, or in the presence ofbodily fluids). Appropriate binders include, but are not limited to,biocompatible polymers and copolymers well known in the art for use insustained release formulations. Such biocompatible compounds arenon-toxic and inert to surrounding tissues, and do not triggersignificant adverse side effects, such as nasal irritation, immuneresponse, inflammation, or the like. They are metabolized into metabolicproducts that are also biocompatible and easily eliminated from thebody.

Exemplary polymeric materials for use in the present disclosure include,but are not limited to, polymeric matrices derived from copolymeric andhomopolymeric polyesters having hydrolyzable ester linkages. A number ofthese are known in the art to be biodegradable and to lead todegradation products having no or low toxicity. Exemplary polymersinclude polyglycolic acids and polylactic acids, poly(DL-lacticacid-co-glycolic acid), poly(D-lactic acid-co-glycolic acid), andpoly(L-lactic acid-co-glycolic acid). Other useful biodegradable orbioerodable polymers include, but are not limited to, such polymers aspoly(epsilon-caprolactone), poly(epsilon-aprolactone-CO-lactic acid),poly(epsilon.-aprolactone-CO-glycolic acid), poly(beta-hydroxy butyricacid), poly(alkyl-2-cyanoacrilate), hydrogels, such as poly(hydroxyethylmethacrylate), polyamides, poly(amino acids) (for example, L-leucine,glutamic acid, L-aspartic acid and the like), poly(ester urea),poly(2-hydroxyethyl DL-aspartamide), polyacetal polymers,polyorthoesters, polycarbonate, polymaleamides, polysaccharides, andcopolymers thereof. Many methods for preparing such formulations arewell known to those skilled in the art (see, for example, Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978). Other useful formulations includecontrolled-release microcapsules (U.S. Pat. Nos. 4,652,441 and4,917,893), lactic acid-glycolic acid copolymers useful in makingmicrocapsules and other formulations (U.S. Pat. Nos. 4,677,191 and4,728,721) and sustained-release compositions for water-soluble peptides(U.S. Pat. No. 4,675,189).

The pharmaceutical compositions of the disclosure typically are sterileand stable under conditions of manufacture, storage and use. Sterilesolutions can be prepared by incorporating the compound in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated herein, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thecompound and/or other biologically active agent into a sterile vehiclethat contains a basic dispersion medium and the required otheringredients from those enumerated herein. In the case of sterilepowders, methods of preparation include vacuum drying and freeze-dryingwhich yields a powder of the compound plus any additional desiredingredient from a previously sterile-filtered solution thereof. Theprevention of the action of microorganisms can be accomplished byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like.

In accordance with the various treatment methods of the disclosure, thecompound can be delivered to a subject in a manner consistent withconventional methodologies associated with management of the disorderfor which treatment or prevention is sought. In accordance with thedisclosure herein, a prophylactically or therapeutically effectiveamount of the compound and/or other biologically active agent isadministered to a subject in need of such treatment for a time and underconditions sufficient to prevent, inhibit, and/or ameliorate a selecteddisease or condition or one or more symptom(s) thereof.

The administration of the compound of the disclosure can be for eitherprophylactic or therapeutic purpose. When provided prophylactically, thecompound is provided in advance of any symptom. The prophylacticadministration of the compound serves to prevent or ameliorate anysubsequent disease process. When provided therapeutically, the compoundis provided at (or shortly after) the onset of a symptom of disease orinfection.

For prophylactic and therapeutic purposes, the compound can beadministered to the subject by the oral route or in a single bolusdelivery, via continuous delivery (for example, continuous transdermal,mucosal or intravenous delivery) over an extended time period, or in arepeated administration protocol (for example, by an hourly, daily orweekly, repeated administration protocol). The therapeutically effectivedosage of the compound can be provided as repeated doses within aprolonged prophylaxis or treatment regimen that will yield clinicallysignificant results to alleviate one or more symptoms or detectableconditions associated with a targeted disease or condition as set forthherein. Determination of effective dosages in this context is typicallybased on animal model studies followed up by human clinical trials andis guided by administration protocols that significantly reduce theoccurrence or severity of targeted disease symptoms or conditions in thesubject. Suitable models in this regard include, for example, murine,rat, avian, porcine, feline, non-human primate, and other acceptedanimal model subjects known in the art. Alternatively, effective dosagescan be determined using in vitro models. Using such models, onlyordinary calculations and adjustments are required to determine anappropriate concentration and dose to administer a therapeuticallyeffective amount of the compound (for example, amounts that areeffective to elicit a desired immune response or alleviate one or moresymptoms of a targeted disease). In alternative embodiments, aneffective amount or effective dose of the compound may simply inhibit orenhance one or more selected biological activities correlated with adisease or condition, as set forth herein, for either therapeutic ordiagnostic purposes.

Radiation Protection and Mitigation

As used herein, any compounds used for prevention, mitigation ortreatment in a subject of injury caused by radiation exposure isadministered in an amount effective to prevent, mitigate or treat suchinjury, namely in an amount and in a dosage regimen effective to preventinjury or to reduce the duration and/or severity of the injury resultingfrom radiation exposure. According to one non-limiting embodiment, aneffective dose ranges from 0.1 or 1 mg/kg to 100 mg/kg, including anyincrement or range therebetween, including 1 mg/kg, 5 mg/kg, 10 mg/kg,20 mg/kg, 25 mg/kg, 50 mg/kg, and 75 mg/kg. However, for each compounddescribed herein, an effective dose or dose range is expected to varyfrom that of other compounds described herein for any number of reasons,including the molecular weight of the compound, bioavailability,specific activity, etc. The therapeutic window between theminimally-effective dose, and maximum tolerable dose in a subject can bedetermined empirically by a person of skill in the art, with end pointsbeing determinable by in vitro and in vivo assays, such as thosedescribed herein and/or are acceptable in the pharmaceutical and medicalarts for obtaining such information regarding radioprotective agents.Different concentrations of the compounds described herein are expectedto achieve similar results, with the drug product administered, forexample and without limitation, once prior to an expected radiationdose, such as prior to radiation therapy or diagnostic exposure toionizing radiation, during exposure to radiation, or after exposure inany effective dosage regimen. The compounds can be administered one ormore times daily, once every two, three, four, five or more days,weekly, monthly, etc., including increments therebetween. A person ofordinary skill in the pharmaceutical and medical arts will appreciatethat it will be a matter of simple design choice and optimization toidentify a suitable dosage regimen for prevention, mitigation ortreatment of injury due to exposure to radiation.

The compounds described herein also are useful in preventing, mitigating(to make less severe) and/or treating injury caused by radiationexposure. In one embodiment, the radiation is ionizing radiation.Ionizing radiation consists of highly-energetic particles or waves thatcan detach (ionize) at least one electron from an atom or molecule.Examples of ionizing radiation are energetic beta particles, neutrons,and alpha particles. The ability of light waves (photons) to ionize anatom or molecule varies across the electromagnetic spectrum. X-rays andgamma rays can ionize almost any molecule or atom; far ultraviolet lightcan ionize many atoms and molecules; near ultraviolet and visible lightare ionizing to very few molecules. Microwaves and radio waves typicallyare considered to be non-ionizing radiation, though damage caused by,e.g., microwaves, may result in the production of free-radicals as partof the injury and/or physiological response to the injury.

Radiotherapy Protection Radiotherapy and Cancer

Radiation therapy works by directing ionizing radiation into the areabeing treated with the goal of damaging the genetic material ofcancerous cells thereby making it impossible for these cells to divide.Accordingly, radiotherapy is an important tool in the fight againstcancer and is used in the treatment of as many as 50% of all cancerpatients. In fact, more than half a million cancer patients receiveradiation therapy each year, either alone or in conjunction withsurgery, chemotherapy or other forms of cancer therapy. Other terms forradiotherapy include radiation therapy, x-ray therapy, electron beamtherapy, cobalt therapy, or irradiation.

Radiotherapy is especially useful in cases where surgical removal of thecancer is not possible, where surgery might debilitate the patient, orwhere surgical debulking of the tumor has not absolutely removed allcancerous tissue. Radiotherapy is routinely used following surgery todestroy any cancer cells that were not removed by surgery. Further usesof radiotherapy are prior to surgery where it can “shrink” a previouslyinoperable tumor down to a manageable size to enable surgical excision.

Radiation therapy can also be used to help relieve symptoms of advancedcancer (such as bleeding or pain), even if a cure is not possible. Overone-third of the practice of radiation therapy is palliative. Thetypical intent of palliative treatment is to relieve pain quickly andmaintain symptom control for the duration of the patient's life.Accordingly, treatment is usually tailored to the patient's clinicalcondition and overall prognosis. Palliative treatment is oftencomplementary to analgesic drug therapies and may enhance theireffectiveness because it can directly target the cause of pain.

Specifically, radiotherapy can be used to treat localized solid tumors,such as cancers of the skin, head and neck, brain, breast, prostate,cervix, and the like. Radiation therapy can also be used to treatcancers of the blood-forming cells and lymphatic system includingleukemia and lymphoma respectively, and the like. Mucous membranes orhair in the vicinity of the radiation or in the path of the radiation(e.g., scalp hair in the case of a brain tumor and rectal mucosa in thecase of prostate cancer) can be protected using the presently disclosedcompounds.

Radiation Forms and Dosage

External beam radiation therapy commonly uses photons, which aresometimes called “packets of energy,” to treat cancer. It is an objectherein to ameliorate the negative effects of all radiotherapy regardlessof the form of the photon or particle, including x-rays, gamma rays, UVrays including UV-A, UV-B and UV-C, neutrons, protons, and electronsincluding beta particles and the like.

X-rays are a very common form of radiation used in radiotherapy. Gammarays are another form of photons used in radiotherapy. Gamma rays can beproduced spontaneously as certain elements (such as radium, uranium, andcobalt 60), which release radiation as they decompose, or decay. Eachelement decays at a specific rate and can give off energy in the form ofgamma rays and other particles. Typically x-rays and gamma rays have thesame general effect on cancer cells.

External beam radiation therapy can be delivered by means of a linearaccelerator. Typically, linear accelerators use powerful generators tocreate the high energy rays for external beam radiation therapy.Generally, linear accelerators are capable of producing x-rays atvarious energies. The linear accelerator can include a special set oflead shutters, called collimators, which focus and direct the rays tothe tumor. The linear accelerator can be a large “L-shaped” design whichallows it to rotate and deliver radiation from all angles. Multipleangles allow the maximum amount of radiation to be delivered to thetumor while delivering a minimal amount of radiation to the surroundinghealthy tissue. The compounds and methods described herein can be usedin conjunction with collimators or other devices and methods that limitradiation exposure to normal cells.

Compounds and methods described herein may be capable of amelioratingthe effects of most forms of radiotherapy. For example, the compoundsand methods can ameliorate the effects of local-field radiation andwide-field radiation. Local field radiation relates to a narrow beam ofradiation directed at the specific metastatic site or sites.Customarily, local field radiation has tended to be used for patientswith a long life expectancy and fewer metastatic sites. In contrast,wide-field radiation employs a larger field of radiation and is oftenused to treat patients with a shorter life expectancy and multiplemetastatic pain-causing sites.

Radiotherapy dosage is measured by the scientific unit rad (radiationabsorbed dose) which is a radiation energy dose equal to an energy of100 ergs per gram of irradiated material. A patient who receivesradiation therapy as a treatment for cancer can receive several thousandrads over a very short period of time (weeks or months). In contrast, atypical scanning x-ray contains far fewer rads. For example, modernmammography systems used to take x-ray images of the breast useapproximately 0.1 to 0.2 rad dose per x-ray.

According to traditional radiotherapy, the larger the daily dose ofradiation, the lower the total dose that can be administered because oflimits to normal tissue tolerance. Proportionately more tumor cells arekilled when the daily radiation dose is larger. Typically a balance isobtained between the killing of tumor cells and the adverse radiationeffects on normal tissues, which are largely a function of the dailydose. A number of different schedules have been developed that take intoaccount specific tumor characteristics and the tolerance of normaltissues. The literature is divided regarding the optimal radiationschedule to achieve tumor regression and disease palliation of eitherprimary or metastatic sites. Generally, however, radiation treatment isplanned in relation to clinical status. Because a main objective hereinis to ameliorate the negative effects of radiation therapy, normaltissue can have a higher tolerance to radiation therapy and largerdosages of radiation can be administered safely.

Side Effects of Radiation

In general, radiation therapy is a local treatment. It typically affectsthe cells in the treated area. However, as mentioned above, in additionto damaging cancer cells, radiation can also damage normal cells locatedin the treated area. Normal cells that are located in the treated areacan include skin cells, mucous membranes, hair follicles, and the like.

Radiation side effects are typically restricted to the radiation portaland can be classified as either acute, occurring during or immediatelyafter the course of radiation therapy, or late, occurring months toyears later. Acute radiation effects are more prominent with radiationschedules that deliver high total doses of radiation with small dailyfractions; they generally begin at the end of the second week oftherapy. Acute radiation effects, occurring primarily at skin andmucosal surfaces, usually consist of an inflammatory response such asskin erythema or pigmentation, or as mucositis. Late radiation effectsmay arise without any preceding acute reactions. Fibrosis is the mostcommon type of late radiation injury and can be observed in many typesof tissue, including skin.

Other skin conditions caused by radiation therapy include dry and moistdesquamation. Dry desquamation, which is characterized by dry and flakyskin and pruritus in the area of irradiation. Moist desquamation, ischaracterized by sloughing of the epidermis, exposing the moist, raw,dermis layer of the skin.

By the phrase protecting from radiation damage it is implied thatrelative to damage expected to be incurred to cells, tissue, or organismwithin a subject or within biological material following exposure to agiven amount of radiation (for example ionizing, infra-red orultra-violet radiation) damage is prevented, minimized or reduced due toeffect of the radioprotector compound.

Clinical radiation sources include beam sources (e.g., X-ray, gammarays, proton beams, etc.) and material sources (e.g., as radium,uranium, cesium 131, cobalt 60, samarium 145, iodine 125 and 127, etc.)that for example may be applied on and/or around a tumor site, orsystemically, parenterally, or orally administered.

In certain embodiments the compounds disclosed herein are administeredpreferentially to cells, tissues or organs likely to be exposed toradiation but that are intended to be protected from such radiationexposure. For example, in the case of administration in conjunction withcancer radiotherapy the formulation will preferably be administeredpreferentially to normal (non-tumor) tissues or cells surrounding atumor or lesion that are likely to be exposed to radiation in the courseof radiotherapy.

In certain embodiments the tumor or neoplasm to be treated is of acancer selected from the group consisting of lung cancer, colorectalcancer, NSCLC, bronchoalveolar cell lung cancer, bone cancer, pancreaticcancer, skin cancer, cancer of the head or neck, cutaneous melanoma,intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer,anal region cancer, stomach cancer, gastric cancer, colon cancer, breastcancer, uterine cancer, fallopian tube carcinoma, endometrial carcinoma,cervical carcinoma, vaginal carcinoma, vulval carcinoma, Hodgkin'sDisease, esophagus cancer, small intestine cancer, endocrine systemcancer, thyroid gland cancer, parathyroid gland cancer, adrenal glandcancer, soft tissue sarcoma, urethral cancer, penis cancer, prostatecancer, bladder cancer, kidney cancer, ureter cancer, renal cellcarcinoma, renal pelvis carcinoma, mesothelioma, hepatocellular cancer,biliary cancer, chronic leukemia, acute leukemia, lymphocytic lymphoma,CNS neoplasm, spinal axis cancer, brain stem glioma, glioblastomamultiform, astrocytoma, schwannoma, ependymoma, medulloblastoma,meningioma, squamous cell carcinoma and pituitary adenoma tumors, andtumor metastasis. In certain embodiments the tumor or tumor metastasisis refractory.

In certain embodiments the radiation is produced by an implantedradiation source and/or by a beam radiation source. In certainembodiments the compound disclosed herein is co-administered with ananti-cancer drug. For example, the radioprotective compounds describedherein can also be used advantageously in therapy in combination withother medicaments, such as chemotherapeutic agents, for example,radiomimetic agents that are cytotoxic agents that cells, tissues,and/or organs in a manner similar to ionizing radiation. Examples ofradiomimetic agents include, but are not limited to bleomycin,doxorubicin, adriamycin, SFU, neocarcinostatin, alkylating agents andother agents that produce DNA adducts.

The compound may be administered prior to radiation exposure. In certainembodiments, the compound may be administered after irradiation, butbefore the appearance of symptoms. In certain embodiments, and compoundsmay be administered after the appearance of symptoms, which may mitigatesymptoms or may treat established complications.

In particular, certain embodiments of the oxetane-substituted compounds(e.g., MMS350) are radiation protector/mitigators that are watersoluble, and easily administered (e.g., orally administered to asubject). The expense of producing MMS350 is far lower thanMnSOD-Plasmid Liposomes, which have been shown to prevent/delayradiation fibrosis in the same mouse model. Furthermore, MnSOD-PL isrelatively ineffective when delivered weeks—

months after irradiation, at the time that fibrosis is forming, andappears to be only effective when delivered prior to irradiation. Incontrast, MMS350 as shown in the data reported herein, has higheffectiveness when delivered months after irradiation and at the time ofinitiation ofradiation fibrosis in the lung. Thus, MMS350 is a novel agent,inexpensive to produce, safe (a toxic dose has not yet been reached indose escalation experiments to date in mice), and it has a unique easeof administration.

In initial experiments, tissue culture using hematopoietic progenitorcell line, IL-3 dependent, 32D cl 3 cells, which forms colonies insemisolid medium showed that MMS350 demonstrated significanteffectiveness as both a radiation protector (delivered beforeirradiation) and radiation damage mitigator (when drug is deliveredafter irradiation). MMS350 was delivered by intraperitoneal injection toC57BL/6 mice (n=15/group) before or after the 9.5 Gy of total bodyirradiation (a dose which kills at least 50% of mice at 30 days) andindicative of death from the hematopoietic syndrome (bone marrowfailure). In this experiment, MMS350 was both a protector when deliveredprior to irradiation and mitigator when delivered after irradiationcomparable to JP4-039. These experiments demonstrate that MMS350 is botha

potent radiation protector and mitigator against total body irradiationand may be of value as a radiation countermeasure against thehematopoietic syndrome.

Additional Therapeutic Indications of Oxetane-Substituted Compounds(Prevention of Fibrosis)

One of the most pressing problems in clinical radiotherapy is defining away to treat the late side effects of irradiation. Acute radiationtoxicity, as an example of the hematopoietic syndrome as describedabove, is treated with agents to alleviate the inflammatory reaction tocell death caused by radiation. After the acute effects of irradiation,there is a latent period during which there is no clinical orhistopathologic signs of irradiation damage. After the latent period,depending on irradiation dose, fraction size, and time over whichirradiation is delivered, as well as volume of tissue treated, the lateside effects of irradiation are initiated. The most prominent lateeffect is fibrosis. A valuable model for measuring effects of newradiation protective or therapeutic agents against the late effects ofirradiation is the lung fibrosis model in C57BL/6 mice. Mice irradiatedto the thoracic cavity (head and neck shielded, abdomen and lower bodyshielded) demonstrate profound radiation fibrosis (organizingalveolitis) at around 120-150 days after thoracic irradiation. In singlefraction experiments delivering 20 Gy to the upper body (thoraciccavity) and in fractionated irradiation experiments, this model offibrosis has been shown to be a valuable test in which to measure theeffectiveness of agents that can protect, mitigate, or treat as atherapeutic (eliminate the fibrosis after it has already formed andthere are signs and symptoms of lung compromise, as a definition of“treatment” in this particular embodiment), and this system has beenused to measure effectiveness of multiple other therapeutic candidateagents.

In a mouse model for radiation fibrosis, a significant component of thefibrotic lesion has been shown to come from bone marrow stromal cells,which migrate through the circulation into the lungs. This model hasbeen published previously and shown to be effective for quantitation ofthe contribution of fibrosis from both resident in situ of fibroblastprogenitors and cells coming from the bone marrow microenvironment, asbone marrow stromal cells. In previous experiments, contribution of thefibrotic lesion in the lung from bone marrow derived cells has beenshown to be significant and range from 20-50%.

MMS350 was tested in two mouse models of fibrosis in the C57BL/6J model.In the first system (chimeric mice) C57BL/6 mice were irradiated to atotal body dose of 10 Gy, which is known to be lethal for 100% ofanimals in the absence of bone marrow transplantation. These mice werethen transplanted intravenously with luciferase positive C57BL/6J mousebone marrow. These mice are valuable for measuring the site ofhematopoiesis from donor origin marrow since the animals can be imagedfor mobilization in a specific imaging system, which detects theluciferase signal. In this assay system, animals are injected withluciferin (the substrate for the enzyme luciferase) bone marrow cells,which contain luciferase (those from the donor marrow strain) metabolizeluciferin and produce a fluorescence which is detected in a camerasystem over the immobilized mice. These same animals can be imagedserially over several weeks—months and followed for migration ofluciferase positive cells from bone marrow sites into irradiated sites.

Chimeric mice with luciferase positive bone marrow demonstrated marrowresistance in bone marrow sites including pelvis, long bones, skull, andtail, but no significant migration of cells into the lungs. Thisirradiation total body dose, which was given to produce chimerism, waswell below the threshold for causing radiation lung fibrosis in thismouse strain, according to previous publications. There was nosignificant migration of cells into the lungs. A subgroup of theseanimals that received an additional boost of irradiation to the thoraciccavity demonstrated significant migration into the chest/lungs ofluciferase positive cells. MMS350 placed in the drinking watersignificantly reduces migration of bone marrow cells into the lungsafter a second thoracic irradiation dose. This result provides strongevidence that MMS350 is preventing radiation fibrosis.

In a second experiment, a clonal bone marrow stromal cell lineestablished from long-term bone marrow cultures of luciferase positivemice and shown to be fluorescent in vitro when luciferin is added to thecells in culture was injected. Cells were injected intraperitoneally inmice that received no thoracic irradiation and in serial photographsover time, the cells stayed in the abdomen. When mice were irradiated tothe thoracic cavity (head and neck and abdomen shielded) to 20 Gy andluciferin positive bone marrow stromal cells were injected into theperitoneal cavity, the luc+ cells migrated into the lungs. The positiveareas were known to be in the lung, because in representative mice,sacrificed and then the heart removed, animals placed back in theimaging system showed identical positivity in the lungs, demonstratingthat the fluorescence from luciferin that was activated by luciferase,were in fact in the lungs. Administration of MMS350 in the drinkingwater to mice in a subgroup of this experiment demonstrated asignificant decrease in migration into the lungs of luc+ cells causingfibrosis. Thus, there is a novel and unique indication for the use ofMMS350, and similar oxetane-substituted compounds as an agent which canprevent radiation fibrosis in irradiated lung, which is susceptible tothis late side effect of irradiation.

Increasing Aqueous Solubility

The oxetane-substituted compounds disclosed herein may also be usefulfor enhancing the dissolution of organic compounds with poor aqueoussolubilities. For example, the oxetane-substituted compounds may bemixed with (i) a cosolvent such as, for example, N-methyl-2-pyrrolidone(NMP), dimethyl sulfoxide (DMSO), alcoholic solvents (e.g., ethanol orisopropyl alcohol), and acetonitrile and (ii) a water-insoluble compoundin an aqueous medium. The resulting composition may also includeadditives such as pH buffers (e.g., borax or a phosphate) that aretypically used in storage compositions.

EXAMPLES Compound Synthesis

A representative synthetic scheme for bifunctional sulfides andsulfones, as well as specific representative examples thereof, are setforth below.

Scheme 1.

Materials and Methods.

General.

Moisture-sensitive reactions were performed under an atmosphere ofnitrogen. 3-Tosyloxymethyl-3-methyl-oxetane was prepared according to aliterature protocol. Curcumin (Acros, 95%), naproxen (Acros, 99%),quinine (Acros, 99%), DMSO (Aldrich, 99.9+%), and HPLC-grade water(Aldrich, CHROMASOLV®) were purchased from commercial suppliers and usedas received. Carbendazim (Aldrich, 97%) was recrystallized from absoluteEtOH, and griseofulvin (Acros, 97%) was recrystallized from toluene.N-Methyl-2-pyrrolidone (Acros, 99%) was distilled from CaH₂ under vacuumand stored over 4 Å MS. All other reagents were used as received unlessotherwise stated. Analytical thin-layer chromatography (TLC) wasperformed on pre-coated silica gel 60 F-254 plates (particle size0.040-0.050 mm, 230-400 mesh) and visualization was accomplished bystaining with KMnO₄ or p-anisaldehyde solutions. ¹H NMR spectra (CDCl₃)and ¹³C NMR spectra (CDCl₃) were referenced to residual chloroform (7.27ppm, ¹H, 77.00 ppm, ¹³C). Chemical shifts (δ) are reported in ppm usingthe following convention: chemical shift, multiplicity (s=singlet,d=doublet, t=triplet, q=quartet, m=multiplet, b=broad), couplingconstants, and integration. IR spectra were collected asattenuated-total-reflection infrared (ATR-IR) spectra. Mass spectra wereobtained on a Micromass Autospec double focusing instrument. UV/VISspectra were recorded on a Perkin Elmer Lambda EZ210 spectrophotometer.pH Determinations were made using a 3 mm Ross™ glass combination micropH electrode (model 8220BNWP) after calibration in standard buffersolutions (pH 4.0, 7.0, and 10.0) at rt.

Bis((3-methyloxetan-3-yl)methyl)sulfane (6)

A 3-necked 3-L round-bottom flask equipped with an overhead stirrer,internal thermometer, and a third arm bearing an argon balloon wascharged with 3-tosyloxymethyl-3-methyl-oxetane 4 (45.4 g, 177 mmol) andbackfilled with N₂ (3×). To the flask was added acetonitrile (900 mL)via cannula. The reaction apparatus was placed in a large heatingmantle. The argon balloon was replaced with a 250-mL addition funnelcontaining a solution of Na₂S.9H₂O (94.5 g 386 mmol) in degassed H₂O(100 mL). The solution was added drop-wise over 25 min. Once theaddition was complete, the reaction mixture was heated to 70° C. over 45min and maintained at 70° C. for 1 h. The mixture was cooled to 20° C.(internal temp), the resulting white precipitate was filtered bygravity, and to the filtrate was added EtOAc (1 L). The resultingprecipitate was removed by aspirator filtration, and the filtrate wasdivided into two 1-L batches. To each batch was added water (500 mL),the layers were separated, and the aqueous portion was extracted withEtOAc (2×200 mL). The combined organic layers were washed with brine(100 mL), and the EtOAc layers from the batches were combined and dried(Na₂SO₄) overnight, filtered, and concentrated. Kugelrohr distillationwas performed on the concentrate. One fraction (T<100° C., 15 Torr) wasdiscarded, and subsequent product collection (140° C. <T<160° C.)yielded a yellow distillate. The distillate was taken up in EtOAc (200mL), washed with water (100 mL) and brine (100 mL), dried (Na2SO4), andconcentrated. Kugelrohr distillation (140° C., 15 Torr) afforded 6 (14.2g, 79%) as a yellow-green oil: IR (ATR) 2956, 2924, 2861, 1450, 1236,973, 829 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 4.47 (d, J=5.7 Hz, 4H), 4.38(d, J=6.0 Hz, 4H), 2.93 (s, 4H), 1.38 (s, 4H); ¹³C NMR (75 MHz, CDCl₃) δ81.9, 43.7, 40.3, 23.0; HRMS (ES) m/z calc for C₁₀H₁₈O₂NaS (M+Na)225.0925, found 225.0908.

3,3′-Sulfinylbis(methylene)bis(3-methyloxetane) (3)

A 1-L round-bottom flask was charged with a solution of 6 (14.9 g, 73.6mmol) in MeOH (240 mL) and cooled to 0° C. A solution of NaIO₄ (16.5 g,77.3 mmol) in water (180 mL) was added via addition funnel over ˜15 min.The ice bath was removed and the slurry was warmed to rt. MeOH (2×50 mL,added 20 min apart) was added, and the mixture was stirred for 12 h atrt. The mixture was filtered through a fritted funnel, and the whiteprecipitate was washed with MeOH. The combined filtrate and washingswere concentrated in vacuo, and the concentrate was coevaporated withtoluene (200 mL). CH₂Cl₂ (400 mL) was added to the residue, followed byMgSO₄. The mixture was filtered, and the filtrate was concentrated invacuo to afford crude 3 (15.82 g) as a yellow solid. To the flaskcontaining the crude solid was added toluene (200 mL), and the slurrywas heated to 60° C. to affect complete dissolution. Decolorizing carbonwas added, and the mixture was filtered by gravity into a 1-L Erlenmeyerflask. To the colorless solution was slowly added distilled hexanes(˜100 mL total) until cloudiness/precipitation occurred. The mixture wasallowed to stand at rt overnight. Upon filtration and drying under highvacuum, 3 (10.49 g) was collected as a white solid. Material recoveredfrom the mother liquor was recrystallized to afford an additional 2.68 gof 3 as white solid for a total yield of 82%. Reported analytical datarefer to that of the first crop: Mp 92.8-94.1° C.; IR (ATR) 2939, 2863,1451, 1381, 1227, 1026, 971 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 4.80 (d,J=6.0 Hz, 2H), 4.61 (d, J=5.6 Hz, 2H), 4.50 (d, J=5.4 Hz, 2H), 4.45 (d,J=6.0 Hz, 2H), 3.38 (d, J=12.9 Hz, 2H), 2.75 (d, J=12.9 Hz, 2H), 1.61(s, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 82.4, 82.0, 61.9, 38.4, 23.4; HRMS(ES) m/z calc for C₁₀H₁₈O₃NaS (M+Na) 241.0874, found 241.0885.

To unambiguously characterize 3 as the sulfoxide, the correspondingsulfone was synthesized from sulfide 6.

3,3′-Sulfonylbis(methylene)bis(3-methyloxetane)

A suspension of oxone (650 mg, 1.06 mmol) in water (2.0 mL) was cooledto 10° C. and treated (dropwise) with a solution of 6 (108 mg, 0.533mmol) in MeOH (2.0 mL). The solution was warmed to rt and stirred for 1h. MeOH was removed in vacuo, and the aqueous layer was diluted withwater (5 mL) and extracted with CH₂Cl₂ (4×10 mL). The combined organiclayers were washed with brine (5 mL), dried (MgSO₄), and concentrated invacuo to afford the sulfone (120 mg, 96%) as a white solid: Mp93.4-95.1° C.; IR (ATR) 2949, 2867, 1456, 1301, 1277, 967 cm⁻¹; ¹H NMR(400 MHz, CDCl₃) δ 4.68 (d, J=6.4 Hz, 4H), 4.46 (d, J=6.4 Hz, 4H), 3.43(s, 4H), 1.69 (s, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 82.2, 62.6, 37.9,23.3; HRMS (APCI) m/z calc for C₁₀H₁₉O₄S (M+H) 235.1004, found 235.1032.

Determination of logP Value of 3.

The logP (octanol-water partition coefficient) was determined using theshake-flask method. Three determinations were made. A representativeprocedure is as follows: a 250-mL reparatory funnel was charged with asolution of 3 (50.0 mg) in water (50.0 mL) and n-octanol (50.0 mL). Thefunnel was capped and inverted 100 times. The funnel and contents wereleft to stand at rt (23.5° C.) for 40 h. Aliquots of both phases wereanalyzed by UV/VIS (214 nm for the aqueous layer and 218 nm for theoctanol layer), and the concentration in each layer was determined usingpreviously generated calibration curves. In the case of the aqueouslayer, a 10-fold dilution was necessary prior to measurement. Allmeasurements were run in triplicate. The logP was determined aslog([3]_(octanol)/[3]_(aqueous)), and the average logP value from the 3trials was −0.87.

Suitable starting materials for the synthetic schemes hereof include,for example:

Examples Increasing Aqueous Solubility

General Procedure for Determination of Solubility in Solutions of 3 andHPLC-Grade Water.

Preparation of Sulfoxide/Water Solutions.

Solutions of 3 and HPLC-grade water were prepared in 1 dram vials bydissolving the appropriate amount of sulfoxide 3 in HPLC-grade water(2.00 mL). In the case of the 25% solution of 3 in H₂O (w/w), 3 (500 mg)was dissolved in water (1.50 mL). Each vial was placed on a platformshaker and shaken at 200 rpm for 30 min.

Solubility Measurements.

Eppendorf vials (1.5 mL size) were charged with model compounds inexcess. Vials were charged with HPLC-grade water (0.500 mL) or theappropriate 3/water solution (0.500 mL). The vials were briefly vortexedand equilibrated in an end-over-end rotator at 30.0° C. for 20 h. Thevials were centrifuged (4000 rpm, 1300×g, 15 min, rt) directly afterremoval from the rotator, and aliquots of the supernatant (0.400 mL)were filtered through 0.45 syringe filters. The pH of each solution wasmeasured using a ThermoScientific electrode (3 mm tip). Each solutionwas diluted with an appropriate volume of either absolute ethanol(quinine, naproxen, griseofulvin) or methanol (carbendazim).Concentrations were calculated by using previously generated calibrationcurves. Appropriate blanks were prepared by diluting aliquots (0.400 mL)of the 3/water solutions (or HPLC-grade water in the case of thecontrol) in an analogous fashion to the sample being measured.

General Procedure for Determination of Solubility in Solutions of DMSOand HPLC-Grade Water.

Preparation of DMSO/Water Solutions.

Solutions of DMSO and HPLC-grade water were prepared in 1 dram vials bydissolving the appropriate amount of DMSO in HPLC-grade water (2.00 mL).Each vial was placed on a platform shaker and shaken at 200 rpm for 30min.

Solubility Measurements.

Eppendorf vials (1.5 mL size) were charged with model compounds inexcess. Vials were charged with either HPLC-grade water (1.00 mL) or theappropriate DMSO/water solution (1.00 mL). The vials were brieflyvortexed and equilibrated in an end-over-end rotator at 30.0° C. for 20h. The vials were centrifuged directly after removal from the rotator(4000 rpm, 1300×g, 15 min, rt), and aliquots of the supernatant (0.800mL) were filtered through 0.45 μm syringe filters. The pH of eachsolution was measured using a ThermoScientific electrode (3 mm tip).Each solution was diluted with an appropriate volume of either absoluteethanol (quinine, naproxen, griseofulvin) or methanol (carbendazim).Concentrations were calculated by using previously generated calibrationcurves. Appropriate blanks were prepared by diluting aliquots (0.800 mL)of the DMSO/water solutions (or HPLC-grade water in the case of thecontrol) in analogous fashion to the sample being measured.

General Procedure for Solubility Determination in Mixtures of Sulfoxide3 in pH 7.0 Buffer.

Preparation of Sulfoxide/Buffer Solutions.

In the case of a 10% w/w solution of sulfoxide 3 in 0.01 M pH 7.0phosphate buffer, 3 (700 mg) was dissolved in 0.01 M Na₂HPO₄/NaH₂PO₄buffer (6.30 mL) and equilibrated on a platform shaker at 200 rpm for 30min. at rt. In the case of a 25% w/w solution of sulfoxide 3 in 0.01 MpH 7.0 phosphate buffer, 3 (1.50 g) was dissolved in 0.01 MNa₂HPO₄/NaH₂PO₄ buffer (4.50 mL) and equilibrated on a platform shakerat 200 rpm for 30 min. at rt.

Solubility Measurements.

Eppendorf vials (1.5 mL size) were charged with model compounds inexcess. Vials were charged with either pH 7.0 phosphate buffer (1.00 mL)or the appropriate 3/buffer solution. The vials were briefly vortexedand equilibrated in an end-over-end rotator at 30.0° C. for 20 h. Thevials were centrifuged directly after removal from the rotator (4000rpm, 1300×g, 15 min, rt), and aliquots of the supernatant (0.800 mL)were filtered through 0.45 μm syringe filters. The pH of each solutionwas measured using a ThermoScientific electrode (3 mm tip) aftercalibration. Each solution was diluted with an appropriate volume ofeither absolute ethanol (quinine, naproxen) or methanol (carbendazim).Concentrations were determined using standard additions of a stocksolution of the compound in either absolute ethanol (quinine, naproxen)or methanol (carbendazim) to aliquots of the diluted filtrates.

General procedure for kinetic solubility measurements. A polypropylenetube was charged with PBS solution (490 μL). To the buffer was added a10 mM stock solution of compound (10 μL). The tube was vortexed andequilibrated on an end-over-end rotator for 15 min at rt. Aliquots (400μL) were filtered through 0.45 μm syringe filters and diluted to 5.0 mLwith absolute MeOH. Concentrations were determined by UV/VIS analysis.

General procedure for brine shrimp toxicity assays. Sample preparation.Stock solutions of 3 were prepared by dissolving 3 (50.0 mg) in 5.0 HPLCgrade water (5.0 mL), (solution A) and 3 (2.50 g) in HPLC grade water(10.0 mL) (solution B). Stock solutions of DMSO were prepared bydiluting DMSO (45 μL) with HPLC grade water (5.0 mL) (solution C) andDMSO (2.27 mL) with HPLC grade water (10.0 mL) (solution D).

In each case, 5 replicates were performed. Each replicate was performedin a 2 dram vial marked at the 4 and 5 mL volume points. To each vialwas added artificial sea water (3 mL) followed by the appropriate volumeof stock solution. For set 1 (1.0 mg/mL), solution A (0.500 mL) orsolution C (0.500 mL) was added. For set 2 (5.0 mg/mL), solution B(0.100 mL) or solution D (0.100 mL) was added to each vial. For set 3(20.0 mg/mL), solution B (0.400 mL) or solution D (0.400 mL) was addedto each vial. For set 4 (50.0 mg/mL), solution B (1.00 mL) or solution D(1.00 mL) was added to each vial. Controls containing HPLC grade water(0.100 mL, 0.400 mL, and 1.00 mL) were prepared in the same manner, andfive replicates of each control were prepared.

Brine Shrimp Hatching.

Brine shrimp eggs (San Francisco Bay Brand) were hatched in a commercialsalt mixture (Instant Ocean). Constant aeration was provided using apump and airstone, and illumination was maintained using a desk lamp.The shrimp were collected in a separate tank after 48 h and used within3-4 h of collection.

Assay.

Brine shrimp were added to each vial using a plastic transfer pipet.After the shrimp were transferred, artificial sea-water was added untilthe volume reached the 5-mL mark. One drop of a yeast suspensionprepared by suspending 11 mg yeast in sea-water (20 mL) was added toeach vial. The shrimp were counted at t=24 h. Another drop of freshlyprepared yeast solution (6 mg in 10 mL sea water) was added, the vialswere maintained under illumination, and shrimp were counted at t=48 h.

General procedure for calculation of water absorption. Oven-dried flaskscapped with septa were cooled under a N₂ atmosphere and charged with avolume of the appropriate solutions (DMSO, 3.0 mL; NMP, 600 μL; 25%3/NMP, 600 μL). The water content was determined by Karl Fischertitration using ˜100 μL aliquots (t=0 measurement). Each septum waspierced with a 1.5 inch 18 gauge needle and left to stand at rt for 7 d.The water content was measured at the end of this period (t=7 dmeasurement) by Karl Fischer titration. All measurements were made induplicate.

Results and Discussion.

Water soluble sulfoxide 3 was prepared in three steps from alcohol 4,which is commercially available or readily prepared from inexpensive2-(hydroxymethyl)-2-methylpropane-1,3-diol. Briefly, treatment of 4 withTsCl in pyridine afforded tosylate 5. Dimeric sulfide formation usingNa₂S followed by oxidation with NaIO₄ provided sulfoxide 3 (Scheme 1).The synthesis is amenable to large-scale preparation and requires nochromatography.

Initially, the aim was to use sulfoxide 3 as a substitute for DMSO inchemical transformations such as modified Swern and Kornblum oxidations.The reasoning was that the resulting sulfide 6, being less volatile andodorous than dimethyl sulfide, would be a more tractable byproduct onindustrial scale. Additionally, bisoxetanyl sulfoxide 3 is watersoluble, and it was found that the sulfide byproduct could be removedfrom reaction mixtures by an oxidative work-up and aqueous washingprocedure. Surprisingly, however, sulfoxide 3 was not a viable oxidizingagent in Kornblum oxidations. For example, α-bromoacetophenone wasstable to an excess of 3 when monitored in CD₃CN over the course ofseveral weeks; under the same conditions, α-bromoacetophenone wasreactive with DMSO within 2 days of treatment.

Taking advantage of the stability and hydrophilicity of 3 that wediscovered, its use for the solubility enhancement of poorly aqueoussoluble compounds was explored. Oxetanes are attractive functionalgroups for increasing aqueous solubility due to their rigid geometriesand exposed polar surface area at the ring ether. Among unsubstitutedcyclic ethers, oxetane has the greatest basicity, which may beattributed to a smaller carbon to oxygen ratio and a larger dipolemoment than its larger counterparts (e.g., THF and THP). Recently,Miller, Carreira, and coworkers demonstrated that replacing agem-dimethyl group with an oxetane moiety can increase a scaffold'saqueous solubility by up to three orders of magnitude while alsoenhancing metabolic stability. We reasoned that sulfoxide 3 may havesufficient aqueous solubility to be completely miscible with water atuseful cosolvent concentrations while also disrupting thehydrogen-bonding network of water, thus aiding in solubilization oflipophilic drug candidate compounds.

To evaluate the utility of sulfoxide 3 as a solubilizing agent, a testset of poorly aqueous soluble compounds, beginning with curcumin 7 waschosen (FIG. 2). Curcumin has shown promise in treating colon cancer andvarious other disorders, but its use is limited in part by its lowaqueous solubility (0.6 μg/mL at ambient temperature, as high as 7.4μg/mL upon heating) and consequently limited bioavailability. Thesolubility of curcumin was measured by UV/VIS spectroscopy afterequilibration for 20 h at ambient temperature in an end-over-endrotator. An increase in aqueous solubility at ambient temperature to60±20 μg/mL using a 25 wt % solution of 3 in water was observed.

The solubility enhancement of 8-12 was further examined by equilibratingthe compounds in solutions of increasing amounts of sulfoxide 3 inwater. Gratifyingly, up to 10-fold increases in aqueous solubility wereobserved for 8-11, and almost 2-fold improvements in aqueous solubilitywere observed in comparison to equivalent DMSO/water solutions (FIGS.3-6).

Based on the solubility curves (FIGS. 3-6), the sulfoxide 3 and DMSOfunction as cosolvents rather than complexing agents. According to themodel derived by Yalkowsky, an exponential increase in observedsolubilities occurs with increasing the volume fraction solventaccording to equation 1, where S_(mix) and S_(w) are the solubility ofthe solute in the cosolvent mixture and water, respectively, σ is thesolubilizing power of the cosolvent, and f_(c) is the volume fraction ofthe cosolvent. The slope of a semi-log plot (σ) is related to thecosolvent's ability to disrupt the intermolecular hydrogen bond networkof water and form a less polar solvent mixture. Thus, the solubilizingpower of sulfoxide 3 and DMSO can be accounted for when comparing thedifference in the experimental logP of sulfoxide 3 (−0.87) and thereported logP of DMSO (−1.3).

Log S _(mix)=log S _(w) +σf _(c)  (1)

Although not bound by any theory, complexation can be tentatively ruledout as a solubilization mechanism assuming that additive-solutecomplexes would form in a 1:1 ratio. If that were the case, a linearcorrelation between additive fraction and solubility would be observed.

The aqueous solubility of estrone 12 is low (0.8 ng/mL), and we wereunable to quantify the aqueous solubility or observe an increase insolubility in the case of a 25% (w/w) mixture of 3 and water; however,increasing the pH of the media using a pH 9.0 buffer (Borax) as well asaddition of NMP to generate a ternary mixture was useful. In this case,the ternary mixture of 3:1:1 pH 9.0 buffer:NMP:DMSO was more effectiveat solubilization than the 3:1:1 pH 9.0 buffer:NMP:3 mixture (Table 1,entries 3 and 4).

TABLE 1 Solubility of estrone 12 in media buffered at pH 9.0. EntryMedium Solubility (μg/mL)^(a) 1 3:1 pH 9.0 buffer/3  30 ± 10 2 3:1 pH9.0 buffer/NMP 63 ± 5 3 3:1:1 pH 9.0 buffer/NMP/3 160 ± 20 4 3:1:1 pH9.0 buffer/NMP/DMSO 230 ± 30 ^(a)Solubility was determined afterequilibration for 20 h at 30.0° C. Data were obtained by UV/VIS analysisof the saturated solutions and were confirmed by analysis ofindependently prepared estrone standards. Each entry represents a meansolubility ± standard deviation (n = 3).

The solubility of test compounds with ionizable functionalities in 0.01M pH 7.0 phosphate buffer was also explored (Table 2). In the case ofnaproxen, the sulfoxide had little effect on the ionized substrate evenat 25% w/w concentrations. The effect on the solubility of quinine wasdiminished at 10% w/w, but an advantage in using 3 was observed in 25%w/w solutions. The solubility of carbendazim in the buffered medium wasthe same as in solutions made from unbuffered HPLC-grade water.

TABLE 2 Solubility of selected model substrates in solutions ofsulfoxide 3 in 0.01M pH 7.0 phosphate buffer. Weight percent 3 inSolution Measured solubility Entry Compound pH 7.0 buffer pH^(a)(μg/mL)^(b) 1 8 0 6.0 1100 (780)^(d)  2 8 10 5.8 1900 (1300)^(d) 3 8 255.2 6200 (3900)^(d) 4 9 0 7.6  950^(c)  5 9 10 8.0 1100  6 9 25 8.21400  7 10 0 6.8 10 8 10 10 6.9 34 9 10 25 7.1 77 ^(a)The pH wasmeasured electrochemically after excess compound was filtered from thesolution. ^(b)Measurements were performed in duplicate unless otherwisenoted. ^(c)Average of 5 trials. ^(d)The two numbers represent data fromseparate trials where entries 1-3 were run in parallel. There was somevariability in the results from the two trials.

The kinetic solubility of two test compounds, carbendazim 10 andgriseofulvin 11, in PBS solution after adding stock solutions preparedin three media: DMSO, NMP, and 25% 3/NMP was also determined. The testcompounds were prepared at concentrations of 10 mM and added to thebuffer at room temperature such that cosolvent concentration was fixedat 2%. The measured solubility was consistent for both test compoundsacross the three stock solutions. While the kinetic solubility ofgriseofulvin was found to be higher than reported (and nearing thethreshold solubility of 200 μM), there was no statistical difference inthe kinetic solubilities of the test compounds among the three differentmedia.

TABLE 3 Kinetic solubility measurements in PBS solutions. Entry CompoundMedium of stock solution Kinetic solubility (μM)^(a) 1 10 DMSO   140 ±13^(b) 2 10 NMP 160 ± 5 3 10 25% 3/NMP 150 ± 9 4 11 DMSO  170 ± 16 5 11NMP  180 ± 13 6 11 25% 3/NMP 190 ± 7 ^(a)Data are reported as mean ±standard deviation (n = 3). ^(b)Data are reported as mean ±standarddeviation (n = 5).

Recently, a series of compounds with nanomolar to micromolar inhibitoryactivity against the serine/threonine protein kinase D (PKD) isoformPKD1 was disclosed (FIG. 7). Several lead structures, especially thosecontaining a pyrimidine moiety, suffered from poor solubility not onlyin aqueous media but also in DMSO. To test the feasibility of usingsulfoxide 3 as a cosolvent for in vitro assays, its use in theradiometric PKD1 inhibition assay was examined. Inhibitory activity attwo concentrations (1 μM and 10 μM) was measured using compound stocksolutions in three formulations: DMSO, NMP, and 25% 3/NMP (FIGS. 8 and9). Comparable biological efficacy was observed for stock solutions in3/NMP vs DMSO. Most significantly, sulfoxide 3 did not interfere withthe standard PKD1 inhibition assay.

Toxicity.

The strain energy of the oxetane ring (25.2 kcal/mol) raises concernsabout the electrophilicity and related toxicity and mutagenicity ofmolecules containing an oxetane moiety. Computational models haveindicated that despite having comparable strain energy to oxirane (26.8kcal/mol), oxetane is 10⁶-times less susceptible to nucleophilicaddition than oxetane. Animal studies have implicated the potentialcarcinogenicity of oxetane and 3,3-dimethyloxetane; it was shown thatboth compounds induce tumor formation at the site of injection in rats.However, a recent report studying the alkylating ability of oxetane,3,3-dimethyloxetane, and 3-methyl-3-oxetanemethanol (1) demonstratedthat these oxetanes are neither mutagenic nor genotoxic. Furthermore,alkylation of NBP 4-(p-nitrobenzyl)pyridine was only observed at acidicpH, implicating that oxetanes do not act as alkylating agents atphysiological pH.

In order to assess the systemic toxicity of oxetane-substitutedsulfoxide 3, a brine shrimp assay was performed (Table 4). Brine shrimpfloating in water containing concentrations of 3 up to 20 mg/mL showed<10% mortality after 48 h. Shrimp incubated in water containing 50 mg/mLof 3 had 85% mortality after 24 h and 100% mortality within 48 h. Thesedata indicate an LC₅₀ of approximately 32 mg/mL (i.e. at 147 mM). Incomparison, brine shrimp treated with DMSO at the same concentrationsshowed no mortality after 24 h and only 15% mortality after 48 h at 50mg/mL of DMSO. These results indicate that sulfoxide 3 may be toleratedin biological assays in concentrations by mass of up to 2%.

TABLE 4 Results from a brine shrimp assay to assess the toxicity ofsulfoxide 3. Concentration % Mortality^(a) % Mortality^(a) EntryCompound (mg/mL) after 24 h after 48 h 1 3 0 <10 10 2 3 1 0 0 3 3 5 0 04 3 20 0-10 0-10 5 3 50 85 100 6 DMSO 0 <10 10 7 DMSO 1 0 0-10 8 DMSO 50 0-6  9 DMSO 20 0 0 10 DMSO 50 0 15 ^(a)Percent mortality wasdetermined for an average of 5 trials. Percent mortality was determinedby estimating the number of shrimp showing no motility after severalminutes of observation.

The cellular toxicity of sulfoxide 3 was further determined for a breastcancer cell line (MDA-MB-231) and a liver cell line (HepG2). In bothcases, the GI₅₀ for 3 was ca. 200 mM, whereas the GI₅₀ for DMSO wasdetermined ca. 800 mM. Interestingly, both cell lines exhibited the samethreshold effect as observed in the case of the brine shrimp, showingonly very limited toxicity at concentrations up to ca. 100 mM.

Because sulfoxide 3 is a solid, it would have to be mixed with anappropriate water-soluble cosolvent to act as a compound storageadditive. NMP was chosen for exploring this potential application due toits thermal stability and low toxicity. Furthermore, it had beendemonstrated that NMP had a greater solubilizing power than ethanol andpropylene glycol, and NMP was previously used for solubilityenhancements both in bioassays and commercial pharmaceuticalapplications such as the Eligard® formulation for delivery of leuprolideto prostate cancer patients. The compound test set (FIG. 2) was storedin 25% w/w solutions of 3 and NMP for 6 weeks at −20° C. During thistime, it was noted that 3 partly precipitated from the solution at thistemperature, but no change in model compound concentration was observedafter thawing of the storage vessels.

A study performed at Abbott indicated that water absorption might inducemore significant compound degradation than oxygen exposure. To ascertainthe degree of water absorption, a 25% w/w 3/NMP solution was monitoredfor one week at ambient temperature (Table 4). Although significantwater absorption was observed (ca. 7,000 ppm over the course of 7 d),the 3/NMP solution absorbed less water than NMP alone. This resultindicates that the hygroscopicity of sulfoxide 3 is low relative to NMP.

TABLE 5 Comparative water absorption measurements of possible compoundstorage media. Water content at t = 0 Water content at t = 7 d EntryMedium (ppm)^(a) (ppm)^(a) 1 DMSO 160, 170^(b) 1080, 1130^(b) 2 NMP 150,130^(c) 12,090, 17,000^(c) 3 25% 3/NMP 160, 200^(c) 6500, 8600^(c) (w/w)^(a)Water content was analyzed by Karl Fischer titration. The two valueslisted represent individual vessels the first value in each columncorresponding to the same vessel at each time-point. ^(b)Average of 3measurements. ^(c)Average of 2 measurements

Conclusion.

The utility of oxetane-substituted sulfoxide 3 as a cosolvent forenhancing the aqueous solubility of model drug compounds wasdemonstrated. Although the relative acute toxicity of 3 was higher thanthat of DMSO in brine shrimp and cell based assays, it was sufficientlylow to permit its use in cellular and in vivo assay development in up to2% final concentrations. Furthermore, sulfoxide 3 proved experimentallyto be far less oxidizing than DMSO, and this property could providegreater stability to long-term compound storage solutions. The amount ofwater absorption will likely depend on the choice of cosolvent, but thenature of 3 (being a solid) may allow the assay developer to choosecosolvents which either do not absorb as much water as DMSO (or NMP) ordo not undergo the dramatic changes in physical properties observed inthe case of wet DMSO solutions. As shown in our PKD1 assays, 3 does notalter the biochemical readout in standard in vitro assays.

This is the first report of the incorporation of an oxetane moiety intoa cosolvent structure for solubility enhancement. The oxetane motifallows for the design of more lipophilic cosolvents that still maintaingood aqueous miscibility due to the dipole moment at the oxetane oxygen.

Examples Radiation Protection Materials and Methods: Mouse Total Bodyand Thoracic Irradiation

C57BL/6TAC mice were obtained from Taconic Farms, and C57BL/6luciferase+ mice were obtained from Steve Thorne, University ofPittsburgh Cancer Institute and housed 5 per cage according toInstitutional IACUC protocols. Total body irradiation andtransplantation of luciferase+ (luc+) marrow was performed

For lung irradiation, mice were irradiated to the thoracic cavity withshielding of the head and neck region and abdomen and lower bodyaccording to published methods. Animals received 20 Gy single fractionthoracic irradiation and were then maintained according to IACUCdirected laboratory conditions. Mice were sacrificed at serial timepoints after thoracic irradiation including pre-irradiation, days 2, 7,14, 28, 50, 75, 100, 110, 150 and 200 post-irradiation. Lungs wereremoved and sagittal sections assayed for percent of lung replaced byfibroblasts and organizing alveolitis according to published methods.Representative lung lobes from the same animals were tested by RT-PCRfor level of detectable message for inflammatory cytokines, redoxsensitive promoters, and levels of MnSOD.

In Vitro Radiation Survival Curves

The murine C57BL/6 bone marrow stromal cell line (Epperly M W, Sikora CA, Defilippi S, Gretton J E, Greenberger J S. Bone marrow origin ofmyofibroblasts in irradiation pulmonary fibrosis. Am J Resp MolecularCell Biology 2003; 29:213-224) and its use in clonogenic radiationsurvival curves has been described (Epperly M W, Gretton J E, BernardingM, Nie S, Rasul B, Greenberger J S. Mitochondrial localization ofcopper/zinc superoxide dismutase (Cu/ZnSOD) confers radioprotectivefunctions in vitro and in vivo. Radiation Research 2003; 160:568-578).

Cell Line and Animal Irradiation

Cell lines were irradiated at dose rate of 70 cGy per minute using aCesium Gamma Cell Irradiator according to published methods. Clonagenicsurvival curve assays with bone marrow stromal cell lines were carriedout according to published methods.

Mice were irradiated using JL Shepherd Mark 1 Model 68 irradiator to9.25 total body irradiation dose or a Varian Linear Accelerator (VarianMedical Systems, Inc, Palo Alto, Calif.) to 20 Gy for pulmonaryirradiation according to published methods (2, 10, 13).

Small Molecule Radiation Protector and Mitigator Drugs

The GS-nitroxide JP4-039 has been described previously (Published U.S.Patent App. US 2010/0035869).

Separation of Different Cellular Components of the Mouse Lung

To isolate different cellular compartments of the lung, mice weresacrificed and the pulmonary cavity was opened, and lungs perfused byinjecting 5 ml of phosphate-buffered saline (PBS) into the rightventricle of the heart. To isolate pulmonary endothelial cells, thelungs were filled with 1 ml of dispase (39.65 mg/ml), allowed tocollapse, and then expanded with 0.5 ml of a 1% low-melt agarose, whichhad been stored at 45° C. in a water bath. The lungs were immediatelycovered with ice and incubated for 2 min. The lungs were then removed,placed in 4 ml of digestion buffer (trypsin 10 ml. HBSS 10 ml, dispase(62.4 mg), and collagenase I (40 mg), incubated for 45 min at 37° C.,and placed on ice. The lungs were transferred to 7 ml of Dulbecco'smodified Eagle's medium (DMEM) containing 0.01% DNA, then teased awayfrom the airways and swirled for 5-10 min at room temperature. Theresulting suspension was filtered through a 40 μm cell strainer,centrifuged at 250 g for 10 min at 4° C., and resuspended in 10 ml ofDMEM. A PE-anti-platelet endothelial cell adhesion molecule (PECAM)monoclonal antibody and an APC-Cy7-anti CD45 monoclonal antibody wereadded to the cells and incubated for 30 mins at 4° C. The cells werewashed in DMEM media, DAPI was added to identify live cells. The cellswere analyzed by flow cytometry with PECAM+ endothelial cells and PECAM−and CD45− cells alveolar cells isolated.

Measurement of Levels of Gene Transcripts for Irradiation InduciblePromoters, Growth Factors, Inflammatory Cytokines, Adhesion MoleculesmicroRNA, and Bromodomain Epigenetic Reader Proteins by RT-PCR

RNA was extracted from mouse lung or purified cell populations using theTRIzol reagent (Invitrogen, Carlsbad, Calif.) following themanufacturer's instructions, quantified using a spectrophotometer, andstored at −80° C. Reverse transcription of 2 μg of total RNA tocomplementary DNA (cDNA) was accomplished using the High Capacity cDNAReverse Transcription Kit (Applied Biosystems, Foster City, Calif.)according to the manufacturer's protocol.

In subsequent steps, expression of GUSB (Gen-Bank: NM_(—)010368.1),NF-KB (Gen-Bank: NM_(—)199267.2), TNF-α (Gen-Bank: NM_(—)013693.2),IFN-γ (Gen-Bank: NM_(—)008337.3), Nrf2 (Gen-bank: NM_(—)010902.3) (21),NF-KB (Gen-Bank: NM_(—)008689.2), JUN (Gen-Bank: NM_(—)010591.2), SP-1(Gen-Bank: NM_(—)013672.2), TGFβ1 (Gen-Bank: NM_(—)011577.1), VEGFa(Gen-Bank: NM_(—)001025250.3), IL-1a (Gen-Bank: NM_(—)010554.4), FGF1(Gen-Bank: NM_(—)010197.3), IFNγ (Gen-Bank: NM_(—)008337.3), IL-6(Gen-Bank: NM_(—)031168.1), FAP (Gen-Bank: NM_(—)007986.2), vWF(Gen-Bank: NM_(—)011708.3), CTGF (Gen-Bank: NM_(—)010217.2) (60), Myd88(Gen-Bank: NM_(—)010851.2), CCL13 (Gen-Bank: NM_(—)018866.2) (61, 67,68), Toll Like Receptors TLR1 (Gen-Bank: NM_(—)030682.1), TLR2(Gen-Bank: NM_(—)011905.3), TLR4 (Gen-Bank: NM_(—)021297.2) (62-63),TLR5 (Gen-Bank: NM_(—)016928.2), TLR6 (Gen-Bank: NM_(—)011604.3), TLR7(Gen-Bank: NM_(—)133211.3), MnSOD (Gen-Bank: NM_(—)013671.3), BMP2(Gen-Bank: NM_(—)007553.2), ADAM12 (Gen-Bank: NM_(—)007400.2), IGFbp7(Gen-Bank: NM_(—)001159518.1), Bromodomain proteins BRD1 (Gen-Bank:NM_(—)001033274.3), BRD2 (Gen-Bank: NM_(—)001204973.1), BRD3 (Gen-Bank:NM_(—)001113573.1), and BRDT (Gen-Bank: NM_(—)001079873.1), and IL-12a(Gen-Bank: NM_(—)001159424.1) was quantitated by real-time polymerasechain reaction (RT-PCR) as described (15). In addition micro-RNA'smi-RNA 107, mi-RNA 126, mi-RNA 155, mi-RNA 511, Let-7d were analyzed forexpression. Ninety-six-well plates were prepared with 10 μl of TaqmanGene Expression Master mix, 5 μl R Nase-free water, 1 μl of thecorresponding Taqman Gene Expression probe, and 4 μl of cDNA (totaling 2μg cDNA) using the Eppendorf epMotion 5070 automated pipetting system(Eppendorf, Westbury, N.Y.). The cDNA was amplified with 40 cycles of95° C. (denaturation) for 15 s and 60° C. (annealing and elongation) for1 min using the Eppendorf Realplex2 Mastercycler.

Data for each gene were normalized by calculating the differences (ΔCt)from the Ct-GUSB and Ct-Target genes. Subsequently, the relativeincrease or decrease in expression was calculated by comparing thereference gene with the target gene (ΔΔCt) and using the formula forrelative expression (=^(2ΔΔCt)). Subsequently, (ΔΔCt) levels werecompared and P values were calculated using one-way ANOVA followed byTukey's multiple comparison tests.

The results were presented as fold increase in RNA above baseline levelswhich were adjusted to that for C57BL/6J/HNsd wild type mice. Thepre-irradiation baseline levels were used to determine the magnitude ofdecrease or elevation in mRNA detectable by robot RT-PCR.

Live Imaging of Luciferase Positive Cells in Marrow and Lungs

C57BL/6NTac mice marrow chimeric luc+ mice were prepared by 9.25 Gy TBI,followed by an injection of 1×10⁷ luc+ marrow cells 24 hours later.Subgroups of these chimeric mice were irradiated to 20 Gy to thepulmonary cavity. On day 75 following thoracic irradiation, 50% of themice were placed on drinking water containing 100 mM MMS350. Mice in thenon-chimeric group were injected intraperitoneally on day 100 afterirradiation with 1×10⁶ luc+ bone marrow stromal cells. Beginning 12 daysafter cell line injection (112 days after irradiation), the cell lineinjected mice were imaged at serial timepoints following injection ofD-luciferin (Gold Biotechnology, St. Louis, Mo.) using a Xenogen IVIS200 Imaging system and the bioluminescent signal for each mousequantitated. As controls for thoracic irradiation, other C57BL/6NTacmice were irradiated to 20 Gy to the right hind limb and injected withluc+ bone marrow stromal cells in the same cell numbers and at the sametime points used for the thoracic irradiation experiment. Animals wereimaged for luc+ cell migration into irradiated hind limbs using methodsidentical to that used for pulmonary irradiation groups. Thebioluminescence of the mice treated with MMS350 in the drinking waterwas compared to mice on regular drinking water using a Student T test.

Pulmonary Histopathology

Lungs from irradiated and luc+ cell line injected mice were removed andserial sectioned. Sections were stained for luc+, GFP+ cells, BrdUincorporation, and for quantitation of percent lung replaced by fibrosis(organizing alveolitis) according to published methods.

Radiation Mitigator Drug Administration

Mice were injected intra-peritoneally with MMS350 or JP4-039(GS-nitroxide) in 50% cremphor/50% ethanol according to publishedmethods. For long-term administration of MMS350, the drug wasadministered in water bottles with bottles changed every 7 days. Thedose of drug per bottle was 100 μM MMS350 starting on day 88. Theestimated total amount consumed by each animal daily was 10.9 μg basedon a mouse drinking 5 ml of water per day. Chimeric mice irradiated tothe thoracic cavity were placed 30 days later on MMS350 in watercontaining 100 mM MMS350. On day 88 after thoracic irradiation ofnon-chimeric mice, mice were placed on 100 mM MMS350 in drinking water.

Results: MMS350 is a Water Soluble Radiation Protector and Mitigator.

MMS350 was protective and mitigative for a C57BL/6 bone marrow stromalcell line cells in vitro (FIG. 12) and mitigated mice against death fromthe hematopoietic syndrome in 9.5 Gy total body irradiated C57BL/6NTacmice (FIG. 13).

Luciferase Bone Marrow Chimeric Mice Demonstrate Pulmonary Migration ofMarrow Cells During the Onset of Fibrosis, which is Ameliorated byMMS350.

The hypothesis that serial time live imaging of luc+ marrow chimericmice would correlate the timing of marrow migration to the lungs, withthe onset of elevation of chronic pulmonary fibrosis related mRNAtranscripts in the lung and endothelial cells, was tested.

Luciferase (luc+) chimeric mice were prepared by total body irradiationaccording to Materials and Methods, and then at day 63 were irradiatedto thoracic cavity to either 18 or 20 Gy according to published methods.

As shown in FIG. 14, total body irradiated, luc+ bone marrow chimericmice demonstrated marrow cavity specific bioluminescence (day 28) and nospecific pulmonary concentration of luc+ cells until 60 days afterthoracic irradiation (day 128). These results were obtained in multipleexperiments. These results confirm and extend experiments with GFP+marrow transplanted mice showing marrow cell migration to the lungs atthe time of onset of pulmonary fibrosis. With luc+ marrow chimeric micethat received 18 Gy or 20 Gy to the thoracic cavity at day 63, subgroupsthat were placed on MMS350 in the drinking water showed reduction ofluc+ areas in the thoracic cavity at day 123 and day 128 post-marrowtransplantation (days 60 and 65 post-thoracic irradiation).

MMS350 Administration Decreases Luciferase+ Bone Marrow Stromal CellHoming and Proliferation in Irradiated Lung.

In a second established experimental model of marrow stromal cell linemigration to the irradiated lung, C57BL/6NTac mice were irradiated to 20Gy to the thoracic cavity and then held for varying intervals. At day 3,60, or 127, subgroups of mice received intraperitoneal injection of2×10⁶ (FIG. 15) cells from a luc+ bone marrow stromal cell line that wasestablished from C57BL6-luc+ GFP+ marrow using published methods. Asshown in FIG. 15, there was no significant accumulation of luc+ cells inthe lungs of mice injected at day 3 (FIGS. 15A, 15D) or day 60 (FIGS.15B, 15E). In marked contrast, mice injected at day 127 showedsignificant migration of luc+ cells to the lungs (FIG. 15C, 15F). Thatthe migration was lung specific was confirmed in mice irradiated to thehind leg and held for equal intervals before injection of the same luc+cell line and showing no cell migration to the irradiated limb (FIG.16). The luc+ cells migrating to the lungs were proliferating in thelung as shown by simultaneous BUDR labeling in luc+ cells (FIG. 17). Theresults were more prominent in mice receiving the higher cell number ofbone marrow stromal cells, also migrating from the peritoneal cavity tothe lungs.

Irradiation Induced Elevation of Pulmonary Gene Transcripts in WholeLung.

Expression of three categories of gene transcripts associated withionizing irradiation effects on the whole lung was initially evaluated.As shown in FIG. 18A, transcripts associated with the acute pulmonaryradiation reaction between days 1 and 14 included inflammatory cytokine(IL-1, TNF-α, TGF-β) and transcripts for promoters associated withoxidative stress (Nrf2), and DNA damage (NFK-B). Transcripts for acutephase associated gene activation products fell after day 14 and remainedlow during the latent period between days 28 and 120. The acuteradiation response phase of the lung has been associated with,pneumonitis related history of pathology including alveolar cells andendothelial cell swelling, alveolar space transudates, and infiltrationwith inflammatory cells. Resolution of the acute phase has beenassociated with a latent period during which the histopathology of thelung returns to normal.

During the latent period, a different pattern of gene transcriptexpression was detected in endothelial cell specific genes. As shown inFIG. 18B, endothelial cell associated gene transcripts remained elevatedduring the latent period (vWF, VEGF, FGF1) as well as CTGF and IL-6.Endothelial cell associated transcripts remained elevated after initialinduction during the acute phase well into the latent period unlikeinflammation associated transcripts (TGF-β, TNF-α, and MnSOD), whichfell to baseline (FIG. 18C).

The late, fibrotic phase of irradiation induced pulmonary damage hasbeen associated with a secondary elevation of some of the gene productsinitially identified during the acute phase. As shown in FIG. 18C,during the late fibrotic phase, after day 120 and extending out to day200, there was elevation in expression of MnSOD, lysl oxidase, TGF-β,IGFbp7, and TLR family genes (prominently TLR4). These data establish apattern of elevated endothelial cell associated gene transcripts duringthe latent period, and suggest ongoing biologic changes in the lungduring a time when histopathologic changes are not identifiable.

Levels of Expression of miRNA Transcripts in Irradiated Lung FollowEstablished Patterns with Induction of RNA Transcripts.

MicroRNA transcripts have been shown to be expressed in reciprocallevels with known patterns of upregulation or downregulation of specificRNA moieties in a regulating manner. The possibility that irradiationfibrosis might be associated with an inappropriate or unsynchronizedupregulation or downregulation of microRNAs was tested. Suchdysregulation could explain the induction of high levels of transcriptsfor gene products associated with fibrosis. miRNAs associated with TLR4(mil07 and 511) (FIG. 19A), vWF (mi126) (FIG. 19B), and with TGF-β(mi155) (FIG. 19C) were evaluated as test cases. MicroRNA Mi107, Mi511,Mi126, and Mi155 responded with levels of RNA for TGF-β, vWF, and TLR4in expected patterns.

Therefore, the observed patterns of upregulation and downregulation ofmiRNAs associated with irradiation-induction of RNA for TLR4, VEGF, andTGF-β were as predicted. The data establish that irradiation alteredmiRNA levels, but could not explain the elevated late phase orpersistent RNA expression patterns observed with TLR4, VEGF, or TGF-β.

Endothelial Cell Specific Upregulation of Gene Transcripts During theLatent Period Between Acute and Chronic Radiation Pulmonary Damage

Populations of cells from the lung at several time points during theacute reaction, latent period, and chronic fibrosis phase, wereseparated. Endothelial cells were compared with alveolar type II cells,epithelial cells, and alveolar macrophages, and separation methods werethose used in previous publications. The results shown in FIG. 20demonstrate elevation in both alveolar and endothelial cells of NFK-B,Nrf2 (FIG. 20A), TLR4, IGFbp7 (FIG. 20B), MnSOD, TGF-β (FIG. 20C), vWF,and VEGF (FIG. 20D). These results establish that the elevations inspecific transcripts include those that were endothelial cell specific(vWF and VEGF) and others seen in both alveolar and endothelial cells(TGF-β, TLR4, NFK-B). The elevation of specific transcripts wassignificantly greater in endothelial cells compared to alveolar cellsfrom the same lungs.

Bromodomain Epigenetic Reader Protein Transcripts are Uniquely ElevatedDuring the Latent Period.

The above results establish that there are clear transcriptionaldifferences between the acute radiation pneumonitis phase, the latentperiod, and the late fibrosis phase in irradiated C57BL/6NTac mice. Theresults establish that endothelial cell specific transcripts werecontinually upregulated during all three phases of the radiationresponse in the lung in whole lung and in separated endothelial comparedto the alveolar cells.

Given that very low levels of intrinsic lung cell divisions occurringprior to the onset of fibrosis (as detectable by BrdU uptake in vivo),we next searched for evidence of genetic and epigenetic changes evolvingin pulmonary endothelial cells, which might explain their role ininitiating migration to the lung of marrow stromal cells during the latefibrotic phase. To attempt to quantitate genetic changes, endothelialcells from lungs at 120 days after thoracic irradiation were explantedand grown in a combination of endothelial specific growth factors (VEGF,FGF1, PPGF). Cell division was monitored and testing for chromosomalaberrations by karyotype analysis of metaphase spreads was sought. Avery low number of endothelial cells removed from irradiated lungattached to plastic or glass surfaces compared to cells fromunirradiated control lungs, and an even lower number went through a celldivision, preventing analysis of metaphases.

Epigenetic changes were quantitated by analysis of the expression ofBromodomain epigenetic reader proteins, as shown in FIG. 21, there was aunique early and late fibrotic phase reduced expression of BRD1, BRD2,BRD3 specifically in lung endothelial cells. In contrast, elevation inthese transcripts during the latent period was detected in separatedendothelial and alveolar cells and in whole lung. As a negative control,testes-specific BRDT protein was not detectable in any pulmonaryendothelial or epithelial lung specimens. These data establish that boththe acute pneumonitis-associated and late fibrosis phase of theradiation pulmonary response in C57BL/6NTac mice includes suppression ofbromodomain epigenetic reader proteins.

MMS350 Reduces Expression of Genes Associated with Late IrradiationFibrosis.

The hypothesis that administration of MMS350 continuously during thetime of induction of gene transcripts associated with the fibrotic phasemight reduce both gene transcription and radiation pulmonary fibrosiswas tested. MMS350 at 100 μM was administered in drinking watercontinuously from day 80, after 20 Gy thoracic irradiation during thelatent period, and throughout the fibrotic period. Mice receiving MMS350showed decreased expression of fibrosis phase mRNA expression (FIG. 19),and decreased associated cytokine and inflammatory marker RNA (FIG. 20),but little effect on bromodomain transcripts, which were already low(FIG. 21). Endothelial and alveolar cell specific expression of fibroticphase associated RNA was reduced by MMS350. These results establish abiomolecular correlation of the effect of the MMS350 mediated decreasein luc+ cell stromal cell migration to the lungs.

Endothelial Cells do not Release Detectable Humoral Factors thatStimulate Stromal Cell Migration In Vitro.

Endothelial cells from late fibrotic phase irradiated lung could bereleasing a humoral factor that stimulated migration of stromal cells tothe lungs through the circulation. Real time tracking/imaging of luc+ BMstromal cell motility in response to endothelial cell and type IIalveolar cell conditioned medium (CM) was performed. Endothelial andtype II alveolar cells were isolated from mouse lung on 150 postirradiation and from unirradiated lung using flow cytometry. Theisolated cells were cultured and CM was harvested after 24 hours. Asshown in FIG. 22, there was no significant effect of adding CM fromexplanted late phase irradiated pulmonary endothelial or alveolar cellson luc+ cell motility in vitro.

MMS350 Reduced Fibrosis in Lungs of Thoracic Irradiated Mice.

To determine whether the MMS350 mediated changes in cell biologic andmolecular biologic properties of late phase irradiation fibrosis alsoresulted in decreased histopathological evidence of fibrosis, wefollowed mice placed on MMS350 in the drinking water daily after day 80.As shown in FIG. 23, there was a significant decrease in pulmonaryfibrosis in mice maintained on MMS350 compared to irradiated controlmice.

The present results establish that a novel water soluble oxetanylsulfoxide has potent radiation mitigation properties against both thetotal body irradiation induced hematopoietic syndrome and thoracicirradiation induced late pulmonary fibrosis. MMS350 was an effectiveradiation mitigator against both the LD 50/30 dose of total bodyirradiation in C57BL/6J/HNsd mice when administered in a single dose 24hours after total body irradiation. MMS350 was also an effectiveradiation protector when administered prior to total body irradiation.Radiation mitigation was comparable to that observed with other smallmolecule radiation protector and mitigator drugs.

Administration of MMS350 in drinking water was effective in decreasingthe severity of late radiation pulmonary fibrosis in 20 Gy thoracicirradiated mice and decreased the magnitude of pulmonary homing to andproliferation of luc+ bone marrow stromal cells, in both a marrowchimera and bone marrow stromal cell line injection. The present dataconfirm and extend previous publications showing marrow origin ofradiation lung fibrosis using clonal green fluorescent protein positivebone marrow stromal cell lines.

The present results also establish that MMS350 reduces severalbiomarkers of radiation pulmonary fibrosis. Administration of MMS350 indrinking water during the latent period between days 14 and 120 afterthoracic irradiation was associated with downregulation of transcriptionof mRNA associated with the late pulmonary fibrotic reaction includingTGF-β, TLR4, and TLR7. Furthermore, explanted, separated populations ofpulmonary endothelial cells from mice irradiated and treated withMMS350, showed modulation of the irradiation effect on mRNA transcripts,with some specific to pulmonary endothelial cells and othersdownregulated in both endothelial and alveolar type-II cells. MicroRNAsknown to be associated with upregulation or downregulation oftranscripts for TGF-β, and TLR4, responded inversely to irradiation andwere also downregulated in mice treated with MMS350. A previouspublication has shown that overexpression of miRNA-29 can reducebleomycin induced fibrosis. The present results provide strong evidencethat MMS350 is an effective radiation mitigator for late pulmonaryfibrosis and acts by a mechanism that does not inappropriately alter thebalance of RNA transcript regulation by specific miRNAs.

Acute and chronic ionizing irradiation induced changes in the lungrepresent an excellent model system in which to define the molecular andphysiologic mechanisms involved during the latent period between theacute and chronic tissue damage responses. Inflammation associatedtranscripts were observed during both acute and fibrotic phases but notduring the latent period. We investigated the molecular and cellularbiomarkers linking the acute and chronic pulmonary irradiation damagephases by real time RT-PCR analysis. Endothelial cell specific levels ofgene transcripts for vWF, VEGF, CCL3, IL6, and CTGF were elevated duringthe latent period and preceding onset of fibrosis. Migration to thelungs of luciferase+ marrow stromal cells was associated with lateelevations of TLR4, TGFβ, and MnSOD all inhibited by drinking wateradministration of MMS350.

The present discovery of an elevation during the latent period of vWFand VEGF as well as other endothelial cell markers indicates thatendothelial cells in the irradiated lung demonstrate persistentelevation in gene expression following ionizing irradiation afterhistopathologic markers of the acute response have subsided.

Coupled with the increased expression of endothelial markers in wholelung was elevated gene transcripts in endothelial cells compared toalveolar cells at all time points as well as increased bromodomainexpression in endothelial cells. This data indicate that irradiationinduced damage leading to lung fibrosis may be mediated through theendothelial cells.

Bromodomain protein transcripts also changed following pulmonaryirradiation. Binding of bormodomains to DNA prevents histonedeacetylation and inhibits DNA transcription. During the acute phase andlate phase, there was decreased bromodomain protein transcriptexpression which correlates with increased gene transcription. Duringthe latent phase, bromodomain expression increased correlated toreduction in some gene expression, but not in endothelial markers ofirradiation damage where expression remained elevated. Irradiation mayhave released a bromodomain inhibitor during the acute and late phases.

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention.

1. A compound having the formula:

wherein X is S, SO or SO₂; one of R¹, R², and R³ is O and the others ofR¹, R² and R³ are independently, the same or different, CH₂, or CR¹³wherein, R¹³ is an alkyl group, an alkenyl group, an alkynyl group, atrialkylsilyl group, or —(CH₂)_(m)OR¹⁵, wherein R¹⁵ is an alkyl group oran aryl group and m is an integer in the range of 1 to 10, and one ofR⁵, R⁶, and R⁷ is O and the others of R⁵, R⁶ and R⁷ are independently,the same or different, CH₂, or CR¹⁴ wherein, R¹⁴ is an alkyl group, analkenyl group, an alkynyl group, a trialkylsilyl group, or—(CH₂)_(n)OR¹⁶, wherein R¹⁶ is an alkyl group or an aryl group and n isan integer in the range of 1 to 10; R⁴ and R⁸ are independently, thesame or different, H, an alkyl group, an alkenyl group, an alkynylgroup, an aryl group, a heteroaryl group, a C₁-C₃ alkoxy group, anaryloxy group, or —(CH₂)_(q)OR¹⁷, wherein R¹⁷ is an alkyl group or anaryl group and q is an integer in the range of 1 to 10, provided that R⁴is not a C₁-C₃ alkoxy group or an aryloxy group when R¹ or R³ is O andR⁸ is not a C₁-C₃ alkoxy group or an aryloxy group when R⁵ or R⁷ is O;R⁹, R¹⁰, R¹¹ and R¹² are independently, the same or different, H, analkyl group, an alkenyl group, an alkynyl group, or an aryl group. 2.The compound of claim 1 wherein R¹³ is a C₁-C₃ alkyl group, a C₂-C₃alkenyl group, a C₂-C₃ alkynyl group, or a trialkylsilyl group and R¹⁴is a C₁-C₃ alkyl group, a C₂-C₃ alkenyl group, a C₂-C₃ alkynyl group, ora trialkylsilyl group.
 3. The compound of claim 1 wherein one of R⁹ andR¹⁰ is H and one of R¹¹ and R¹² is H.
 4. The compound of claim 1 whereinone of R¹, R², and R³ is O and the others of R¹, R² and R³ are CH₂, andone of R⁵, R⁶, and R⁷ is O and the others of R⁵, R⁶ and R⁷ are CH₂. 5.The compound of claim 1 wherein X is SO.
 6. The compound of claim 1wherein the compound has the formula:


7. The compound of claim 1 wherein R² and R⁶ are each O; and R¹, R³, R⁵,R⁷ are each CH₂.
 8. The compound of claim 1 wherein R² and R⁶ are eachO; R¹, R³, R⁵, R⁷ are each CH₂; R⁴ and R⁸ are each C₁-C₁₀ alkyl such asa methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl, pentyl,3-pentyl, or hexyl; and R⁹-R¹² are each H.
 9. The compound of claim 1wherein R⁹-R¹² are each H.
 10. The compound of claim 1 wherein X is SO;R² and R⁶ are each O; R¹, R³, R⁵, R⁷ are each CH₂; R⁴ and R⁸ are eachC₁-C₁₀ alkyl such as a methyl, ethyl, propyl, isopropyl, butyl,iso-butyl, sec-butyl, pentyl, 3-pentyl, or hexyl; and R⁹-R¹² are each H.11. The compound of claim 1 wherein the compound has the formula


12. A compound having the formula:

wherein X is S, SO or SO₂; one of R¹, R², and R³ is NR⁶⁰ and the othersof R¹, R² and R³ are independently, the same or different, CH₂, or CR¹³wherein, R¹³ is an alkyl group, an alkenyl group, an alkynyl group, atrialkylsilyl group, or —(CH₂)_(m)OR¹⁵, wherein R¹⁵ is an alkyl group oran aryl group and m is an integer in the range of 1 to 10; one of R⁵,R⁶, and R⁷ is NR⁶¹ and the others of R⁵, R⁶ and R⁷ are independently,the same or different, CH₂, or CR¹⁴ wherein, R¹⁴ is an alkyl group, analkenyl group, an alkynyl group, a trialkylsilyl group, or—(CH₂)_(n)OR¹⁶, wherein R¹⁶ is an alkyl group or an aryl group and n isan integer in the range of 1 to 10, wherein R⁶⁰ and R⁶¹ are eachindependently H, an alkyl group, an alkenyl group, an alkynyl group, anaryl group or a heteroaryl group; R⁴ and R⁸ are independently, the sameor different, H, an alkyl group, an alkenyl group, an alkynyl group, anaryl group, a heteroaryl group, a C₁-C₃ alkoxy group, an aryloxy group,or —(CH₂)_(q)OR¹⁷, wherein R¹⁷ is an alkyl group or an aryl group and qis an integer in the range of 1 to 10; and R⁹, R¹⁰, R¹¹ and R¹² areindependently, the same or different, H, an alkyl group, an alkenylgroup, an alkynyl group, or an aryl group.
 13. The compound of claim 12wherein R² is NR⁶⁰, R⁶ is NR⁶¹, and R¹, R³, R⁵ and R⁷ are each CH₂. 14.The compound of claim 12 wherein R⁶⁰ and R⁶¹ are each a substituted arylgroup.
 15. The compound of claim 12 wherein X is SO; R² is NR⁶⁰, R⁶ isNR⁶¹; R¹, R³, R⁵, R⁷ are each CH₂; R⁴ and R⁸ are each C₁-C₁₀ alkyl suchas a methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl,pentyl, 3-pentyl, or hexyl; and R⁹-R¹² are each H.
 16. A compound havinga formula of:

wherein X is S, SO or SO₂; one of R¹, R², and R³ is O and the others ofR¹, R² and R³ are independently, the same or different, CH₂, or CR¹³wherein, R¹³ is an alkyl group, an alkenyl group, an alkynyl group, atrialkylsilyl group, or —(CH₂)_(m)OR¹⁵, wherein R¹⁵ is an alkyl group oran aryl group and m is an integer in the range of 1 to 10; R⁴ is H, analkyl group, an alkenyl group, an alkynyl group, an aryl group, aheteroaryl group, a C₁-C₃ alkoxy group, an aryloxy group, or—(CH₂)_(q)OR¹⁷, wherein R¹⁷ is an alkyl group or an aryl group and q isan integer in the range of 1 to 10; and R⁹, R¹⁰, R¹¹ and R¹² areindependently, the same or different, H, an alkyl group, an alkenylgroup, an alkynyl group, an aryl group.
 17. The compound of claim 16wherein X is SO; is O; R¹ and R³ are each CH₂; R⁴ is C₁-C₁₀ alkyl suchas a methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl,pentyl, 3-pentyl, or hexyl; and R⁹-R¹² are each H.
 18. A compound havinga formula of:

wherein R³³ includes an oxetanyl sulfane, oxetanyl sulfinyl, or oxetanylsulfonyl moiety; X is one of

R³¹, R³² and R³⁴ are, independently, hydrogen, C₁-C₆ straight orbranched-chain alkyl, optionally including a phenyl (C₆H₅) group, thatoptionally is methyl-, hydroxyl- or fluoro-substituted, including:methyl, ethyl, propyl, 2-propyl, butyl, t-butyl, pentyl, hexyl, benzyl,hydroxybenzyl (e.g., 4-hydroxybenzyl), phenyl and hydroxyphenyl; and R³⁰is —NH—R³⁵, —O—R³⁵ or —CH₂—R³⁵, where R³⁵ is an —N—O., —N—OH or N═Ocontaining group.
 19. The compound of claim 18, wherein the compound hasa structure of:


20. A compound having a formula of:

wherein R⁴⁰ includes an oxetanyl sulfane, oxetanyl sulfinyl, or oxetanylsulfonyl moiety; R⁴¹, R^(41a), R⁴⁴, and R^(44a) are each independentlyhydrogen, a halo, C₁-C₆ straight or branched-chain alkyl, or a C₁-C₆straight or branched-chain alkyl further comprising a phenyl (C₆H₅)group, wherein the C₁-C₆ straight or branched-chain alkyl group or theC₁-C₆ straight or branched-chain alkyl group comprising a phenyl groupis unsubstituted or is methyl-, hydroxyl- or halo-substituted; R⁴ ishydrogen, a halo, a C₁-C₆ straight or branched-chain alkyl, or a C₁-C₆straight or branched-chain alkyl further comprising a phenyl (C₆H₅)group, wherein the C₁-C₆ straight or branched-chain alkyl group or theC₁-C₆ straight or branched-chain alkyl group comprising a phenyl groupis unsubstituted or is methyl-, hydroxyl- or halo-substituted; R⁴⁵ is an—N—O., —N—OH or N═O containing group; R⁴², R⁴³, R⁴⁶, and R^(46a) areindependently H or a halo, a C₁-C₆ straight or branched-chain alkyl, ora C₁-C₆ straight or branched-chain alkyl further comprising a phenyl(C₆H₅) group, wherein the C₁-C₆ straight or branched-chain alkyl groupor the C₁-C₆ straight or branched-chain alkyl group comprising a phenylgroup is unsubstituted or is methyl-, hydroxyl- or halo-substituted. 21.A compound having a formula of:

wherein R²² includes an oxetanyl sulfane, oxetanyl sulfinyl, or oxetanylsulfonyl moiety; R²³, R^(23a), R²¹, and R^(21a) are each independentlyhydrogen, a halo, C₁-C₆ straight or branched-chain alkyl, or a C₁-C₆straight or branched-chain alkyl further comprising a phenyl (C₆H₅)group, wherein the C1-C₆ straight or branched-chain alkyl group or theC₁-C₆ straight or branched-chain alkyl group comprising a phenyl groupis unsubstituted or is methyl-, hydroxyl- or halo-substituted; R⁴ ishydrogen, a halo, a C₁-C₆ straight or branched-chain alkyl, or a C₁-C₆straight or branched-chain alkyl further comprising a phenyl (C₆H₅)group, wherein the C₁-C₆ straight or branched-chain alkyl group or theC₁-C₆ straight or branched-chain alkyl group comprising a phenyl groupis unsubstituted or is methyl-, hydroxyl- or halo-substituted; and R²⁰is an —N—O., —N—OH or N═O containing group.
 22. The compound of claim21, wherein the compound has a structure of:


23. A method of effecting radioprotection, comprising: administering atleast one oxetane-substituted compound.
 24. The method of claim 23,wherein the oxetane-substituted compound is administered to a subject ina therapeutically effective amount.
 25. The method of claim 24, whereinthe oxetane-substituted compound is administered to the subject prior toirradiation of the subject.
 26. (canceled)
 27. A method of effectingradiomitigation, comprising: administering at least oneoxetane-substituted compound.
 28. The method of claim 27 wherein theoxetane-substituted compound is administered to a subject in atherapeutically effective amount.
 29. The method of claim 28 wherein theoxetane-substituted compound is administered to the subject afterirradiation of the subject. 30.-32. (canceled)
 33. The method of claim23 wherein the oxetane-substituted compound is a bifunctional sulfoxide,sulfide or sulfone.
 34. (canceled)
 35. The method of claim 23 whereinthe oxetane-substituted compound has the formula

wherein X is S, SO or SO₂; one of R¹, R², and R³ is O and the others ofR¹, R² and R³ are independently, the same or different, CH₂, or CR¹³wherein, R¹³ is an alkyl group, an alkenyl group, an alkynyl group, atrialkylsilyl group, or —(CH₂)_(m)OR¹⁵, wherein R¹⁵ is an alkyl group oran aryl group and m is an integer in the range of 1 to 10, and one ofR⁵, R⁶, and R⁷ is O and the others of R⁵, R⁶ and R⁷ are independently,the same or different, CH₂, or CR¹⁴ wherein, R¹⁴ is an alkyl group, analkenyl group, an alkynyl group, a trialkylsilyl group, or—(CH₂)_(n)OR¹⁶, wherein R¹⁶ is an alkyl group or an aryl group and n isan integer in the range of 1 to 10; R⁴ and R⁸ are independently, thesame or different, H, an alkyl group, an alkenyl group, an alkynylgroup, an aryl group, a heteroaryl group, a C₁-C₃ alkoxy group, anaryloxy group, or —(CH₂)_(q)OR¹⁷, wherein R¹⁷ is an alkyl group or anaryl group and q is an integer in the range of 1 to 10, provided that R⁴is not a C₁-C₃ alkoxy group or an aryloxy group when R¹ or R³ is O andR⁸ is not a C₁-C₃ alkoxy group or an aryloxy group when R⁵ or R⁷ is O;R⁹, R¹⁰, R¹¹ and R¹² are independently, the same or different, H, analkyl group, an alkenyl group, an alkynyl group, or an aryl group.36.-45. (canceled)
 46. The method of claim 23 wherein theoxetane-substituted compound has the formula

wherein X is S, SO or SO₂; one of R¹, R², and R³ is O and the others ofR¹, R² and R³ are independently, the same or different, CH₂, or CR¹³wherein, R¹³ is an alkyl group, an alkenyl group, an alkynyl group, atrialkylsilyl group, or —(CH₂)_(m)OR¹⁵, wherein R¹⁵ is an alkyl group oran aryl group and m is an integer in the range of 1 to 10; R⁴ is H, analkyl group, an alkenyl group, an alkynyl group, an aryl group, aheteroaryl group, a C₁-C₃ alkoxy group, an aryloxy group, or—(CH₂)_(q)OR¹⁷, wherein R¹⁷ is an alkyl group or an aryl group and q isan integer in the range of 1 to 10; and R⁹, R¹⁰, R¹¹ and R¹² areindependently, the same or different, H, an alkyl group, an alkenylgroup, an alkynyl group, an aryl group.
 47. The method of claim 23wherein the oxetane-substituted compound has the formula

wherein R³³ includes an oxetanyl sulfane, oxetanyl sulfinyl, or oxetanylsulfonyl moiety; X is one of

R³¹, R³² and R³⁴ are, independently, hydrogen, C₁-C₆ straight orbranched-chain alkyl, optionally including a phenyl (C₆H₅) group, thatoptionally is methyl-, hydroxyl- or fluoro-substituted, including:methyl, ethyl, propyl, 2-propyl, butyl, t-butyl, pentyl, hexyl, benzyl,hydroxybenzyl (e.g., 4-hydroxybenzyl), phenyl and hydroxyphenyl; and R³⁰is —NH—R³⁵, —O—R³⁵ or —CH₂—R³⁵, where R³⁵ is an —N—O., —N—OH or N═Ocontaining group.
 48. The method of claim 23 wherein theoxetane-substituted compound has the formula:

wherein R⁴⁰ includes an oxetanyl sulfane, oxetanyl sulfinyl, or oxetanylsulfonyl moiety; R⁴¹, R^(41a), R⁴⁴, and R^(44a) are each independentlyhydrogen, a halo, C₁-C₆ straight or branched-chain alkyl, or a C₁-C₆straight or branched-chain alkyl further comprising a phenyl (C₆H₅)group, wherein the C₁-C₆ straight or branched-chain alkyl group or theC1-C₆ straight or branched-chain alkyl group comprising a phenyl groupis unsubstituted or is methyl-, hydroxyl- or halo-substituted; R₄ ishydrogen, a halo, a C₁-C₆ straight or branched-chain alkyl, or a C₁-C₆straight or branched-chain alkyl further comprising a phenyl (C₆H₅)group, wherein the C₁-C₆ straight or branched-chain alkyl group or theC₁-C₆ straight or branched-chain alkyl group comprising a phenyl groupis unsubstituted or is methyl-, hydroxyl- or halo-substituted; R⁴⁵ is an—N—O., —N—OH or N═O containing group; R⁴², R⁴³, R⁴⁶, and R^(46a) areindependently H or a halo, a C₁-C₆ straight or branched-chain alkyl, ora C₁-C₆ straight or branched-chain alkyl further comprising a phenyl(C₆H₅) group, wherein the C₁-C₆ straight or branched-chain alkyl groupor the C₁-C₆ straight or branched-chain alkyl group comprising a phenylgroup is unsubstituted or is methyl-, hydroxyl- or halo-substituted. 49.The method of claim 23 wherein the oxetane-substituted compound has theformula:

wherein R²² includes an oxetanyl sulfane, oxetanyl sulfinyl, or oxetanylsulfonyl moiety; R²³, R^(23a), R²¹, and R^(21a) are each independentlyhydrogen, a halo, C₁-C₆ straight or branched-chain alkyl, or a C₁-C₆straight or branched-chain alkyl further comprising a phenyl (C₆H₅)group, wherein the C₁-C₆ straight or branched-chain alkyl group or theC₁-C₆ straight or branched-chain alkyl group comprising a phenyl groupis unsubstituted or is methyl-, hydroxyl- or halo-substituted; R⁴ ishydrogen, a halo, a C₁-C₆ straight or branched-chain alkyl, or a C₁-C₆straight or branched-chain alkyl further comprising a phenyl (C₆H₅)group, wherein the C₁-C₆ straight or branched-chain alkyl group or theC₁-C₆ straight or branched-chain alkyl group comprising a phenyl groupis unsubstituted or is methyl-, hydroxyl- or halo-substituted; and R²⁰is an —N—O., —N—OH or N═O containing group.
 50. A method of increasingaqueous solubility of an organic compound, comprising: adding at leastone oxetane-substituted sulfoxide.
 51. A method of increasing solubilityof an organic compound in an aqueous medium, comprising: adding acompound that includes an oxetane moiety. 52.-56. (canceled)
 57. Acomposition comprising the compound of claim 1, and a water solublecosolvent.
 58. (canceled)